Herbicide resistance genes for resistance to aryloxyalkanoate herbicides

ABSTRACT

The subject invention provides novel plants that are not only resistant to 2,4-D, but also to a pyridyloxyacetate herbicide. The subject invention also includes plants that produce one or more enzymes of the subject invention “stacked” together with one or more other herbicide resistance genes. The subject invention enables novel combinations of herbicides to be used in new ways. Furthermore, the subject invention provides novel methods of preventing the development of, and controlling, strains of weeds that are resistant to one or more herbicides such as glyphosate. The preferred enzyme and gene for use according to the subject invention are referred to herein as AAD-13 (AryloxyAlkanoate Dioxygenase). This highly novel discovery is the basis of significant herbicide tolerant crop trait and selectable marker opportunities.

This application is a National Stage filing of PCT InternationalApplication Ser. No. PCT/US2008/63212, filed May 9, 2008, which claimsthe benefit of U.S. Provisional Application Ser. No. 60/928,303, filedMay 9, 2007, the disclosures each of which are expressly incorporatedherein by reference.

BACKGROUND OF THE INVENTION

Weeds can quickly deplete soil of valuable nutrients needed by crops andother desirable plants. There are many different types of herbicidespresently used for the control of weeds. One extremely popular herbicideis glyphosate.

Crops, such as corn, soybeans, canola, cotton, sugar beets, wheat, turf,and rice, have been developed that are resistant to glyphosate. Thus,fields with actively growing glyphosate resistant soybeans, for example,can be sprayed to control weeds without significantly damaging thesoybean plants.

With the introduction of genetically engineered, glyphosate tolerantcrops (GTCs) in the mid-1990's, growers were enabled with a simple,convenient, flexible, and inexpensive tool for controlling a widespectrum of broadleaf and grass weeds unparalleled in agriculture.Consequently, producers were quick to adopt GTCs and in many instancesabandon many of the accepted best agronomic practices such as croprotation, herbicide mode of action rotation, tank mixing, incorporationof mechanical with chemical and cultural weed control. Currentlyglyphosate tolerant soybean, cotton, corn, and canola are commerciallyavailable in the United States and elsewhere in the Western Hemisphere.Alfalfa was the first perennial GTC introduced, furthering theopportunity for repeated use of glyphosate on the same crop and fieldsrepeatedly over a period of years. More GTCs (e.g., wheat, rice, sugarbeets, turf, etc.) are poised for introduction pending global marketacceptance. Many other glyphosate resistant species are in experimentalto development stages (e.g., sugar cane, sunflower, beets, peas, carrot,cucumber, lettuce, onion, strawberry, tomato, and tobacco; forestryspecies like poplar and sweetgum; and horticultural species likemarigold, petunia, and begonias; see “isb.vt.edu/cfdocs/fieldtests1.cfm,2005” website). Additionally, the cost of glyphosate has droppeddramatically in recent years to the point that few conventional weedcontrol programs can effectively compete on price and performance withglyphosate GTC systems.

Glyphosate has been used successfully in burndown and other non-cropareas for total vegetation control for more than 15 years. In manyinstances, as with GTCs, glyphosate has been used 1-3 times per year for3, 5, 10, up to 15 years in a row. These circumstances have led to anover-reliance on glyphosate and GTC technology and have placed a heavyselection pressure on native weed species for plants that are naturallymore tolerant to glyphosate or which have developed a mechanism toresist glyphosate's herbicidal activity.

Extensive use of glyphosate-only weed control programs is resulting inthe selection of glyphosate-resistant weeds, and is selecting for thepropagation of weed species that are inherently more tolerant toglyphosate than most target species (i.e., weed shifts). (Powles andPreston, 2006, Ng et al., 2003; Simarmata et al., 2003; Lorraine-Colwillet al., 2003; Sfiligoj, 2004; Miller et al., 2003; Heap, 2005; Murphy etal., 2002; Martin et al., 2002.) Although glyphosate has been widelyused globally for more than 15 years, only a handful of weeds have beenreported to have developed resistance to glyphosate (Heap, 2005);however, most of these have been identified in the past five years.Resistant weeds include both grass and broadleaf species—Lolium rigidum,Lolium multiflorum, Eleusine indica, Sorghum halepense, Ambrosiaartemisiifolia, Conyza canadensis, Conyza bonariensis, Plantagolanceolata, Amaranthus palmerii, and Amaranthus rudis. Additionally,weeds that had previously not been an agronomic problem prior to thewide use of GTCs are now becoming more prevalent and difficult tocontrol in the context of GTCs, which comprise >80% of U.S. cotton andsoybean acres and >20% of U.S. corn acres (Gianessi, 2005). These weedshifts are occurring predominantly with (but not exclusively)difficult-to-control broadleaf weeds. Some examples include Ipomoea,Amaranthus, Chenopodium, Taraxacum, and Commelina species.

In areas where growers are faced with glyphosate resistant weeds or ashift to more difficult-to-control weed species, growers can compensatefor glyphosate's weaknesses by tank mixing or alternating with otherherbicides that will control the missed weeds. One popular andefficacious tankmix partner for controlling broadleaf escapes in manyinstances has been 2,4-dichlorophenoxyacetic acid (2,4-D). 2,4-D hasbeen used agronomically and in non-crop situations for broad spectrum,broadleaf weed control for more than 60 years. Individual cases of moretolerant species have been reported, but 2,4-D remains one of the mostwidely used herbicides globally. A limitation to further use of 2,4-D isthat its selectivity in dicot crops like soybean or cotton is very poor,and hence 2,4-D is not typically used on (and generally not near)sensitive dicot crops. Additionally, 2,4-D's use in grass crops issomewhat limited by the nature of crop injury that can occur. 2,4-D incombination with glyphosate has been used to provide a more robustburndown treatment prior to planting no-till soybeans and cotton;however, due to these dicot species' sensitivity to 2,4-D, theseburndown treatments must occur at least 14-30 days prior to planting(Agriliance, 2005).

2,4-D is in the phenoxy acid class of herbicides, as is MCPA. 2,4-D hasbeen used in many monocot crops (such as corn, wheat, and rice) for theselective control of broadleaf weeds without severely damaging thedesired crop plants. 2,4-D is a synthetic auxin derivative that acts toderegulate normal cell-hormone homeostasis and impede balanced,controlled growth; however, the exact mode of action is still not known.Triclopyr and fluoroxypyr are pyridyloxyacetic acid herbicides whosemode of action is as a synthetic auxin, also.

These herbicides have different levels of selectivity on certain plants(e.g., dicots are more sensitive than grasses). Differential metabolismby different plants is one explanation for varying levels ofselectivity. In general, plants metabolize 2,4-D slowly, so varyingplant response to 2,4-D may be more likely explained by differentactivity at the target site(s) (WSSA, 2002). Plant metabolism of 2,4-Dtypically occurs via a two-phase mechanism, typically hydroxylationfollowed by conjugation with amino acids or glucose (WSSA, 2002).

Over time, microbial populations have developed an alternative andefficient pathway for degradation of this particular xenobiotic, whichresults in the complete mineralization of 2,4-D. Successive applicationsof the herbicide select for microbes that can utilize the herbicide as acarbon source for growth, giving them a competitive advantage in thesoil. For this reason, 2,4-D currently formulated has a relatively shortsoil half-life, and no significant carryover effects to subsequent cropsare encountered. This adds to the herbicidal utility of 2,4-D.

One organism that has been extensively researched for its ability todegrade 2,4-D is Ralstonia eutropha (Streber et al., 1987). The genethat codes for the first enzymatic step in the mineralization pathway istfdA. See U.S. Pat. No. 6,153,401 and GENBANK Acc. No. M16730. TfdAcatalyzes the conversion of 2,4-D acid to dichlorophenol (DCP) via anα-ketoglutarate-dependent dioxygenase reaction (Smejkal et al., 2001).DCP has little herbicidal activity compared to 2,4-D. TfdA has been usedin transgenic plants to impart 2,4-D resistance in dicot plants (e.g.,cotton and tobacco) normally sensitive to 2,4-D (Streber et al. (1989),Lyon et al. (1989), Lyon (1993), and U.S. Pat. No. 5,608,147).

A large number of tfdA-type genes that encode proteins capable ofdegrading 2,4-D have been identified from the environment and depositedinto the Genbank database. Many homologues are similar to tfdA (>85%amino acid identity) and have similar enzymatic properties to tfdA.However, there are a number of homologues that have a significantlylower identity to tfdA (25-50%), yet have the characteristic residuesassociated with α-ketoglutarate dioxygenase Fe⁺² dioxygenases. It istherefore not obvious what the substrate specificities of thesedivergent dioxygenases are.

One unique example with low homology to tfdA (35% amino acid identity)is sdpA from Sphingobium herbicidovorans (Kohler et al., 1999,Westendorf et al., 2002, Westendorf et al., 2003). This enzyme has beenshown to catalyze the first step in (S)-dichlorprop (and other(S)-phenoxypropionic acids) as well as 2,4-D (a phenoxyacetic acid)mineralization (Westendorf et al., 2003). Transformation of this geneinto plants has not heretofore been reported.

Development of new herbicide-tolerant crop (HTC) technologies has beenlimited in success due largely to the efficacy, low cost, andconvenience of GTCs. Consequently, a very high rate of adoption for GTCshas occurred among producers. This created little incentive fordeveloping new HTC technologies.

Aryloxyalkanoate chemical substructures are a common entity of manycommercialized herbicides including the phenoxyacetate auxins (such as2,4-D and dichlorprop), pyridyloxyacetate auxins (such as fluoroxypyrand triclopyr), aryloxyphenoxypropionates (AOPP) acetyl-coenzyme Acarboxylase (ACCase) inhibitors (such as haloxyfop, quizalofop, anddiclofop), and 5-substituted phenoxyacetate protoporphyrinogen oxidaseIX inhibitors (such as pyraflufen and flumiclorac). However, theseclasses of herbicides are all quite distinct, and no evidence exists inthe current literature for common degradation pathways among thesechemical classes. A multifunctional enzyme for the degradation ofherbicides covering multiple modes of action has recently been described(PCT US/2005/014737; filed May 2, 2005). Another unique multifunctionalenzyme and potential uses are described hereafter.

BRIEF SUMMARY OF THE INVENTION

The subject invention provides novel plants that are not only resistantto 2,4-D, but also to pyridyloxyacetate herbicides. Heretofore, therewas no expectation or suggestion that a plant with both of theseadvantageous properties could be produced by the introduction of asingle gene. The subject invention also includes plants that produce oneor more enzymes of the subject invention “stacked” together with one ormore other herbicide resistance genes, including, but not limited to,glyphosate-, ALS- (imidazolinone, sulfonylurea), aryloxyalkanoate-,HPPD-, PPO-, and glufosinate-resistance genes, so as to provideherbicide-tolerant plants compatible with broader and more robust weedcontrol and herbicide resistance management options. The presentinvention further includes methods and compositions utilizing homologuesof the genes and proteins exemplified herein.

In some embodiments, the invention provides monocot and dicot plantstolerant to 2,4-D, MCPA fluoroxypyr, and one or more commerciallyavailable herbicides (e.g., glyphosate, glufosinate, paraquat,ALS-inhibitors (e.g., sulfonylureas, imidazolinones, triazolopyrimidinesulfonanilides, et al), HPPD inhibitors (e.g, mesotrione, isoxaflutole,et al.), dicamba, bromoxynil, aryloxyphenoxypropionates, and others).Vectors comprising nucleic acid sequences responsible for such herbicidetolerance are also disclosed, as are methods of using such tolerantplants and combinations of herbicides for weed control and prevention ofweed population shifts. The subject invention enables novel combinationsof herbicides to be used in new ways. Furthermore, the subject inventionprovides novel methods of preventing the development of, andcontrolling, strains of weeds that are resistant to one or moreherbicides such as glyphosate. The subject invention enables novel usesof novel combinations of herbicides and crops, including preplantapplication to an area to be planted immediately prior to planting withseed for plants that would otherwise be sensitive to that herbicide(such as 2,4-D).

The subject invention relates in part to the identification of an enzymethat is not only able to degrade 2,4-D, but also surprisingly possessesnovel properties, which distinguish the enzyme of the subject inventionfrom previously known tfdA-type proteins, for example. Morespecifically, the subject invention relates to the use of an enzyme thatis capable of degrading both 2,4-D and pyridyloxyacetate herbicides. Noα-ketoglutarate-dependent dioxygenase enzyme has previously beenreported to have the ability to degrade herbicides of both thephenoxyacetate and pyridyloxyacetates auxin herbicides. The preferredenzyme and gene for use according to the subject invention are referredto herein as AAD-13 (AryloxyAlkanoate Dioxygenase). This highly noveldiscovery is the basis of significant herbicide-tolerant crop (HTC)trait and selectable marker opportunities. Plants of the subjectinvention can be resistant throughout their entire life cycle.

There was no prior motivation to produce plants comprising an AAD-13gene (preferably an AAD-13 polynucleotide that has a sequence optimizedfor expression in one or more types of plants, as exemplified herein),and there was no expectation that such plants could effectively producean AAD-13 enzyme to render the plants resistant a phenoxyacetic acidherbicide (such as 2,4-D) and/or one or more pyridyloxyacetatesherbicides such as triclopyr and fluoroxypyr. Thus, the subjectinvention provides many advantages that were not heretofore thought tobe possible in the art.

This invention also relates in part to the identification and use ofgenes encoding aryloxyalkanoate dioxygenase enzymes that are capable ofdegrading phenoxyacetate auxin and/or pyridyloxyacetates auxinherbicides. Methods of screening proteins for these activities arewithin the scope of the subject invention. Thus, the subject inventionincludes degradation of 2,4-dichlorophenoxyacetic acid and otheraryloxyalkanoate auxin herbicides by a recombinantly expressed AAD-13enzyme. The subject invention also includes methods of controlling weedswherein said methods comprise applying one or more pyridyloxyacetate orphenoxyacetate auxin herbicides to plants comprising an AAD-13 gene. Thesubject invention also provides methods of using an AAD-13 gene as aselectable marker for identifying plant cells and whole plantstransformed with AAD-13, optionally including one, two, or moreexogenous genes simultaneously inserted into target plant cells. Methodsof the subject invention include selecting transformed cells that areresistant to appropriate levels of an herbicide. The subject inventionfurther includes methods of preparing a polypeptide, having thebiological activity of aryloxyalkanoate dioxygenase, by culturing plantsand/or cells of the subject invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the general chemical reaction that is catalyzed byAAD-13 enzymes of the subject invention.

FIG. 2 is a ClustalW alignment of α-ketoglutarate dioxygenases. Residuesconserved in 80% of the sequences are highlighted. (Identical andsimilar residues are highlighted.) SEQ ID NO:2 provides the sequence ofthe aad 13 sequence in FIG. 2. SEQ ID NO:12 provides the sequence of theaad 13 sequence in FIG. 2. SEQ ID NO:13 provides the sequence of the aadsequence in FIG. 2. SEQ ID NO:14 provides the sequence of the aad 2sequence in FIG. 2. SEQ ID NO:15 provides the sequence of the tfdAsequence in FIG. 2. SEQ ID NO:16 provides the sequence of the tauDsequence in FIG. 2.

FIG. 3 illustrates the concomitant breakdown of α-ketoglutarate and thesubstrate of interest via AAD-13.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the native nucleotide sequence of AAD-13 from Sphingobiumherbicidovorans.

SEQ ID NO:2 is the translated protein sequence encoded by SEQ ID NO:1.

SEQ ID NO:3 is the plant optimized nucleotide sequence of AAD-13 (v1).

SEQ ID NO:4 is the translated protein sequence encoded by SEQ ID NO:3.

SEQ ID NO:5 is the E. coli optimized nucleotide sequence of AAD-13 (v2).

SEQ ID NO:6 shows the sequence of the “sdpacodF” AAD-13 (v1) primer.

SEQ ID NO:7 shows the sequence of the “sdpacodR” AAD-13 (v1) primer.

SEQ ID NO:8 shows the sequence of the “sucCD” primer.

SEQ ID NO:9 shows the sequence of the “sucCD” primer.

SEQ ID NO:10 shows the sequence of the AAD-13 (v2) primer.

SEQ ID NO:11 shows the sequence of the AAD-13 (v2) primer.

SEQ ID NO:12 shows the sequence of aad 12 as in FIG. 2.

SEQ ID NO:13 shows the sequence of aad 1 as in FIG. 2.

SEQ ID NO:14 shows the sequence of aad 2 as in FIG. 2.

SEQ ID NO:15 shows the sequence of tfdA as in FIG. 2.

SEQ ID NO:16 shows the sequence of tauD as in FIG. 2.

DETAILED DESCRIPTION OF THE INVENTION

The subject development of a 2,4-D resistance gene and subsequentresistant crops provides excellent options for controlling broadleaf,glyphosate-resistant (or highly tolerant and shifted) weed species forin-crop applications. 2,4-D is a broad-spectrum, relatively inexpensive,and robust broadleaf herbicide that would provide excellent utility forgrowers if greater crop tolerance could be provided in dicot and monocotcrops alike. 2,4-D-tolerant transgenic dicot crops would also havegreater flexibility in the timing and rate of application. An additionalutility of the subject herbicide tolerance trait for 2,4-D is itsutility to prevent damage to normally sensitive crops from 2,4-D drift,volatilization, inversion (or other off-site movement phenomenon),misapplication, vandalism, and the like. An additional benefit of theAAD-13 gene is that unlike all tfdA homologues characterized to date,AAD-13 is able to degrade the pyridyloxyacetates auxin (e.g.,fluoroxypyr) in addition to achiral phenoxy auxins (e.g., 2,4-D, MCPA,4-chlorophenoxyacetic acid). See Table 1. A general illustration of thechemical reactions catalyzed by the subject AAD-13 enzyme is shown inFIG. 1. (Addition of O₂ is stereospecific; breakdown of intermediate tophenol and glyoxylate is spontaneous.) It should be understood that thechemical structures in FIG. 1 illustrate the molecular backbones andthat various R groups and the like (such as those shown in Table 1) areincluded but are not necessarily specifically illustrated in FIG. 1.Multiple mixes of different phenoxy auxin combinations have been usedglobally to address specific weed spectra and environmental conditionsin various regions. Use of the AAD-13 gene in plants affords protectionto a much wider spectrum of auxin herbicides, thereby increasing theflexibility and spectra of weeds that can be controlled. The subjectinvention can also be used to protect from drift or other off-sitesynthetic auxin herbicide injury for the full breadth of commerciallyavailable phenoxy auxins. Table 1 defines commercially availablepyridyloxy and phenoxy auxins and provides relevant chemical structures.

TABLE 1 Commercially available phenoxyacetate and pyridiyloxyacetateauxins. Reference to phenoxy auxin and pyridyloxy auxin herbicides isgenerally made to the active acid but some are commercially formulatedas any of a variety of corresponding ester formulations and these arelikewise considered as substrates for AAD-13 enzyme in planta as generalplant esterases convert these sters to the active acids in planta.Likewise reference can also be for the corresponding organic orinorganic salt of the corresponding acid. Possible use rate ranges canbe as stand- alone treatments or in combination with other herbicides inboth crop and non- crop uses. Preferred Possible use use rate Chemicalrate ranges ranges name CAS no (g ae/ha) (g ae/ha) Structure 2,4-D  94-75-7 25-4000 280-1120

2,4,5-T   93-76-5 25-4000  25-4000

4-CPA  122-88-3 25-4000  25-4000

3,4-DA  588-22-7 25-4000  25-4000

MCPA   94-76-6 25-4000 125-1550

Triclopyr 55335-06-3 50-2000  70-840

Fluroxypyr 69377-81-7 25-2000  35-560

A single gene (AAD-13) has now been identified which, when geneticallyengineered for expression in plants, has the properties to allow the useof phenoxy auxin herbicides in plants where inherent tolerance neverexisted or was not sufficiently high to allow use of these herbicides.Additionally, AAD-13 can provide protection in planta topyridyloxyacetate herbicides where natural tolerance also was notsufficient to allow selectivity, expanding the potential utility ofthese herbicides. Plants containing AAD-13 alone now may be treatedsequentially or tank mixed with one, two, or a combination of severalphenoxy auxin herbicides. The rate for each phenoxy auxin herbicide mayrange from 25 to 4000 g ae/ha, and more typically from 100 to 2000 gae/ha for the control of a broad spectrum of dicot weeds. Likewise, one,two, or a mixture of several pyridyloxyacetate auxin compounds may beapplied to plants expressing AAD-13 with reduced risk of injury fromsaid herbicides. The rate for each pyridyloxyacetate herbicide may rangefrom 25 to 2000 g ae/ha, and more typically from 35-840 g ae/ha for thecontrol of additional dicot weeds.

Glyphosate is used extensively because it controls a very wide spectrumof broadleaf and grass weed species. However, repeated use of glyphosatein GTCs and in non-crop applications has, and will continue to, selectfor weed shifts to naturally more tolerant species orglyphosate-resistant biotypes. Tankmix herbicide partners used atefficacious rates that offer control of the same species but havingdifferent modes of action is prescribed by most herbicide resistancemanagement strategies as a method to delay the appearance of resistantweeds. Stacking AAD-13 with a glyphosate tolerance trait (and/or withother herbicide-tolerance traits) could provide a mechanism to allow forthe control of glyphosate resistant dicot weed species in GTCs byenabling the use of glyphosate, phenoxy auxin(s) (e.g., 2,4-D) andpyridyloxyacetates auxin herbicides (e.g., fluoroxypyr)—selectively inthe same crop. Applications of these herbicides could be simultaneouslyin a tank mixture comprising two or more herbicides of different modesof action; individual applications of single herbicide composition insequential applications as pre-plant, preemergence, or postemergence andsplit timing of applications ranging from approximately 2 hours toapproximately 3 months; or, alternatively, any combination of any numberof herbicides representing each chemical class can be applied at anytiming within about 7 months of planting the crop up to harvest of thecrop (or the preharvest interval for the individual herbicide, whicheveris shortest).

It is important to have flexibility in controlling a broad spectrum ofgrass and broadleaf weeds in terms of timing of application, rate ofindividual herbicides, and the ability to control difficult or resistantweeds. Glyphosate applications in a crop with a glyphosate resistancegene/AAD-13 stack could range from about 250-2500 g ae/ha; phenoxy auxinherbicide(s) (one or more) could be applied from about 25-4000 g ae/ha;and pyridyloxyacetates auxin herbicide(s) (one or more) could be appliedfrom 25-2000 g ae/ha. The optimal combination(s) and timing of theseapplication(s) will depend on the particular situation, species, andenvironment, and will be best determined by a person skilled in the artof weed control and having the benefit of the subject disclosure.

Plantlets are typically resistant throughout the entire growing cycle.Transformed plants will typically be resistant to new herbicideapplication at any time the gene is expressed. Tolerance is shown hereinto 2,4-D across the life cycle using the constitutive promoters testedthus far (primarily CsVMV and AtUbi 10). One would typically expectthis, but it is an improvement upon other non-metabolic activities wheretolerance can be significantly impacted by the reduced expression of asite of action mechanism of resistance, for example. One example isRoundup Ready cotton, where the plants were tolerant if sprayed early,but if sprayed too late the glyphosate concentrated in the meristems(because it is not metabolized and is translocated); viral promotersMonsanto used are not well expressed in the flowers. The subjectinvention provides an improvement in these regards.

Herbicide formulations (e.g., ester, acid, or salt formulation; orsoluble concentrate, emulsifiable concentrate, or soluble liquid) andtankmix additives (e.g., adjuvants, surfactants, drift retardants, orcompatibility agents) can significantly affect weed control from a givenherbicide or combination of one or more herbicides. Any combination ofthese with any of the aforementioned herbicide chemistries is within thescope of this invention.

One skilled in the art would also see the benefit of combining two ormore modes of action for increasing the spectrum of weeds controlledand/or for the control of naturally more tolerant or resistant weedspecies. This could also extend to chemistries for which herbicidetolerance was enabled in crops through human involvement (eithertransgenically or non-transgenically) beyond GTCs. Indeed, traitsencoding glyphosate resistance (e.g., resistant plant or bacterial EPSPS(including Agro. strain CP4), glyphosate oxidoreductase (GOX), GAT),glufosinate resistance (e.g., Pat, bar), acetolactate synthase(ALS)-inhibiting herbicide resistance (e.g., imidazolinone,sulfonylurea, triazolopyrimidine sulfonanilide, pyrmidinylthiobenzoates,and other chemistries=AHAS, CsrI, SurA, et al.), bromoxynil resistance(e.g., Bxn), resistance to inhibitors of HPPD(4-hydroxlphenyl-pyruvate-dioxygenase) enzyme, resistance to inhibitorsof phytoene desaturase (PDS), resistance to photosystem II inhibitingherbicides (e.g., psbA), resistance to photosystem I inhibitingherbicides, resistance to protoporphyrinogen oxidase IX (PPO)-inhibitingherbicides (e.g., PPO-1), resistance to phenylurea herbicides (e.g.,CYP76B1), dicamba-degrading enzymes (see, e.g., US 20030135879), andothers could be stacked alone or in multiple combinations to provide theability to effectively control or prevent weed shifts and/or resistanceto any herbicide of the aforementioned classes. In vivo modified EPSPScan be used in some preferred embodiments, as well as Class I, Class II,and Class III glyphosate resistance genes.

Regarding additional herbicides, some additional preferred ALSinhibitors include but are not limited to the sulfonylureas (such aschlorsulfuron, halosulfuron, nicosulfuron, sulfometuron, sulfosulfuron,trifloxysulfuron), imidazoloninones (such as imazamox, imazethapyr,imazaquin), triazolopyrimidine sulfonanilides (such ascloransulam-methyl, diclosulam, florasulam, flumetsulam, metosulam, andpenoxsulam), pyrimidinylthiobenzoates (such as bispyribac andpyrithiobac), and flucarbazone. Some preferred HPPD inhibitors includebut are not limited to mesotrione, isoxaflutole, and sulcotrione. Somepreferred PPO inhibitors include but are not limited to flumiclorac,flumioxazin, flufenpyr, pyraflufen, fluthiacet, butafenacil,carfentrazone, sulfentrazone, and the diphenylethers (such asacifluorfen, fomesafen, lactofen, and oxyfluorfen).

Additionally, AAD-13 alone or stacked with one or more additional HTCtraits can be stacked with one or more additional input (e.g., insectresistance, fungal resistance, or stress tolerance, et al.) or output(e.g., increased yield, improved oil profile, improved fiber quality, etal.) traits. Thus, the subject invention can be used to provide acomplete agronomic package of improved crop quality with the ability toflexibly and cost effectively control any number of agronomic pests.

The subject invention relates in part to the identification of an enzymethat is not only able to degrade 2,4-D, but also surprisingly possessesnovel properties, which distinguish the enzyme of the subject inventionfrom previously known tfdA proteins, for example. Even though thisenzyme has very low homology to tfdA, the genes of the subject inventioncan still be generally classified in the same overall family ofα-ketoglutarate-dependent dioxygenases. This family of proteins ischaracterized by three conserved histidine residues in a“HX(D/E)X₂₃₋₂₆(T/S)X₁₁₄₋₁₈₃HX₁₀₋₁₃R” motif which comprises the activesite. The histidines coordinate Fe⁺² ion in the active site that isessential for catalytic activity (Hogan et al., 2000). The preliminaryin vitro expression experiments discussed herein were tailored to helpselect for novel attributes. These experiments also indicate the AAD-13enzyme is unique from another disparate enzyme of the same class,disclosed in a previously filed patent application (PCT US/2005/014737;filed May 2, 2005). The AAD-1 enzyme of that application shares onlyabout 25% sequence identity with the subject AAD-13 protein.

More specifically, the subject invention relates in part to the use ofan enzyme that is not only capable of degrading 2,4-D, but alsopyridyloxyacetate herbicides. No α-ketoglutarate-dependent dioxygenaseenzyme, besides the previously identified AAD-1 and AAD-12 enzymes(subject of patent applications PCT US/2005/014737 (WO 2005/107437) andWO 2007/053482, respectively), has previously been reported to have theability to degrade herbicides of different chemical classes withdifferent modes of action. Preferred enzymes and genes for use accordingto the subject invention are referred to herein as AAD-13(AryloxyAlkanoate Dioxygenase) genes and proteins.

This invention also relates in part to the identification and use ofgenes encoding aryloxyalkanoate dioxygenase enzymes that are capable ofdegrading phenoxy auxin and pyridyloxyacetate herbicides. Thus, thesubject invention relates in part to the degradation of2,4-dichlorophenoxyacetic acid, other phenoxyacetic acids, andpyridyloxyacetic acid herbicides by a recombinantly expressed AAD-13enzyme.

The subject proteins tested positive for 2,4-D conversion to2,4-dichlorophenol (“DCP”; herbicidally inactive) in analytical assays.Partially purified proteins of the subject invention can rapidly convert2,4-D to DCP in vitro. An additional advantage that AAD-13 transformedplants provide is that parent herbicide(s) are metabolized to inactiveforms, thereby reducing the potential for harvesting herbicidal residuesin grain or stover.

The subject invention also includes methods of controlling weeds whereinsaid methods comprise applying a pyridyloxyacetate and/or a phenoxyauxin herbicide to plants comprising an AAD-13 gene.

In light of these discoveries, novel plants that comprise apolynucleotide encoding this type of enzyme are now provided.Heretofore, there was no motivation to produce such plants, and therewas no expectation that such plants could effectively produce thisenzyme to render the plants resistant to not only phenoxy acidherbicides (such as 2,4-D) but also pyridyloxyacetate herbicides. Thus,the subject invention provides many advantages that were not heretoforethought to be possible in the art.

Publicly available strains (deposited in culture collections like ATCCor DSMZ) can be acquired and screened, using techniques disclosedherein, for novel genes. Sequences disclosed herein can be used toamplify and clone the homologous genes into a recombinant expressionsystem for further screening and testing according to the subjectinvention.

As discussed above in the Background section, one organism that has beenextensively researched for its ability to degrade 2,4-D is Ralstoniaeutropha (Streber et al., 1987). The gene that codes for the firstenzyme in the degradation pathway is tfdA. See U.S. Pat. No. 6,153,401and GENBANK Ace. No. M16730. TfdA catalyzes the conversion of 2,4-D acidto herbicidally inactive DCP via an α-ketoglutarate-dependentdioxygenase reaction (Smejkal et al., 2001). TfdA has been used intransgenic plants to impart 2,4-D resistance in dicot plants (e.g.,cotton and tobacco) normally sensitive to 2,4-D (Streber et al., 1989;Lyon et al., 1989; Lyon et al., 1993). A large number of tfdA-type genesthat encode proteins capable of degrading 2,4-D have been identifiedfrom the environment and deposited into the NCBI database. Manyhomologues are quite similar to tfdA (>85% amino acid identity) and havesimilar enzymatic properties to tfdA. However, a small collection ofα-ketoglutarate-dependent dioxygenase homologues are presentlyidentified that have a low level of homology to tfdA.

The subject invention relates in part to surprising discoveries of newuses for and functions of a distantly related enzyme, sdpA, fromSphingobium herbicidovorans (Westendorf et al., 2002, 2003) with lowhomology to tfdA (35% amino acid identity) and low homology to therecently-identified AAD-1 (27% amino acid identity). Thisα-ketoglutarate-dependent dioxygenase enzyme purified in its native formhad previously been shown to degrade 2,4-D and S-dichlorprop (Westendorfet al., 2002 and 2003). However, no α-ketoglutarate-dependentdioxygenase enzyme has previously been reported to have the ability todegrade a selective herbicide of the pyridyloxyacetate chemical class.SdpA (from Sphingobium herbicidovorans) has never been expressed inplants, nor was there any motivation to do so in part becausedevelopment of new HTC technologies has been limited due largely to theefficacy, low cost, and convenience of GTCs (Devine, 2005).

In light of the novel activity, proteins and genes of the subjectinvention are referred to herein as AAD-13 proteins and genes. AAD-13was presently confirmed to degrade a variety of phenoxyacetate auxinherbicides in vitro. See Table 5.4.4-1 in Example 5, below.Additionally, this enzyme, as reported for the first time herein, wassurprisingly found to also be capable of degrading additional substratesof the class of aryloxyalkanoate molecules. Substrates of significantagronomic importance include the pyridyloxyacetate auxin herbicides.This highly novel discovery is the basis of significant HerbicideTolerant Crop (HTC) and selectable marker trait opportunities. Thisenzyme is unique in its ability to deliver herbicide degradativeactivity to a range of broad spectrum broadleaf herbicides(phenoxyacetate and pyridyloxyacetate auxins).

Thus, the subject invention relates in part to the degradation of2,4-dichlorophenoxyacetic acid, other phenoxyacetic auxin herbicides,and pyridyloxyacetate herbicides by a recombinantly expressedaryloxyalkanoate dioxygenase enzyme (AAD-13). This invention alsorelates in part to identification and uses of genes encoding anaryloxyalkanoate dioxygenase degrading enzyme (AAD-13) capable ofdegrading phenoxy and/or pyridyloxy auxin herbicides.

The subject enzyme enables transgenic expression resulting in toleranceto combinations of herbicides that would control nearly all broadleafweeds. AAD-13 can serve as an excellent herbicide tolerant crop (HTC)trait to stack with other HTC traits [e.g., glyphosate resistance,glufosinate resistance, ALS-inhibitor (e.g., imidazolinone,sulfonylurea, triazolopyrimidine sulfonanilide) resistance, bromoxynilresistance, HPPD-inhibitor resistance, PPO-inhibitor resistance, etal.], and insect resistance traits (Cry1F, Cry1Ab, Cry 34/45, other Bt.Proteins, or insecticidal proteins of a non-Bacillis origin, et al.) forexample. Additionally, AAD-13 can serve as a selectable marker to aid inselection of primary transformants of plants genetically engineered witha second gene or group of genes.

In addition, the subject microbial gene has been redesigned such thatthe protein is encoded by codons having a bias toward both monocot anddicot plant usage (hemicot). Arabidopsis, corn, tobacco, cotton,soybean, canola, and rice have been transformed with AAD-13-containingconstructs and have demonstrated high levels of resistance to both thephenoxy and pyridyloxy auxin herbicides. Thus, the subject inventionalso relates to “plant optimized” genes that encode proteins of thesubject invention.

Oxyalkanoate groups are useful for introducing a stable acidfunctionality into herbicides. The acidic group can impart phloemmobility by “acid trapping,” a desirable attribute for herbicide actionand therefore could be incorporated into new herbicides for mobilitypurposes. Aspects of the subject invention also provide a mechanism ofcreating HTCs. There exist many potential commercial and experimentalherbicides that can serve as substrates for AAD-13. Thus, the use of thesubject genes can also result in herbicide tolerance to those otherherbicides as well.

HTC traits of the subject invention can be used in novel combinationswith other HTC traits (including but not limited to glyphosatetolerance). These combinations of traits give rise to novel methods ofcontrolling weed (and like) species, due to the newly acquiredresistance or inherent tolerance to herbicides (e.g., glyphosate). Thus,in addition to the HTC traits, novel methods for controlling weeds usingherbicides, for which herbicide tolerance was created by said enzyme intransgenic crops, are within the scope of the invention.

This invention can be applied in the context of commercializing a 2,4-Dresistance trait stacked with current glyphosate resistance traits insoybeans, for example. Thus, this invention provides a tool to combatbroadleaf weed species shifts and/or selection of herbicide resistantbroadleaf weeds, which culminates from extremely high reliance bygrowers on glyphosate for weed control with various crops.

The transgenic expression of the subject AAD-13 gene is exemplified in,for example, Arabidopsis and tobacco. Soybeans are a preferred crop fortransformation according to the subject invention. However, thisinvention can be utilized in multiple other monocot (such as pasturegrasses or turf grass) and dicot crops like alfalfa, clover, treespecies, et al. Likewise, 2,4-D (or other AAD-13-substrates) can be morepositively utilized in grass crops where tolerance is moderate, andincreased tolerance via this trait would provide growers the opportunityto use these herbicides at more efficacious rates and over a widerapplication timing without the risk of crop injury.

Still further, the subject invention provides a single gene that canprovide resistance to herbicides that control broadleaf weed. This genemay be utilized in multiple crops to enable the use of a broad spectrumherbicide combination. The subject invention can also control weedsresistant to current chemicals, and aids in the control of shifting weedspectra resulting from current agronomic practices. The subject AAD-13can also be used in efforts to effectively detoxify additional herbicidesubstrates to non-herbicidal forms. Thus, the subject invention providesfor the development of additional HTC traits and/or selectable markertechnology.

Separate from, or in addition to, using the subject genes to produceHTCs, the subject genes can also be used as selectable markers forsuccessfully selecting transformants in cell cultures, greenhouses, andin the field. There is high inherent value for the subject genes simplyas a selectable marker for biotechnology projects. The promiscuity ofAAD-13 for other aryloxyalkanoate auxinic herbicides provides manyopportunities to utilize this gene for HTC and/or selectable markerpurposes.

Proteins (and source isolates) of the subject invention. The presentinvention provides functional proteins. By “functional activity” (or“active”) it is meant herein that the proteins/enzymes for use accordingto the subject invention have the ability to degrade or diminish theactivity of a herbicide (alone or in combination with other proteins).Plants producing proteins of the subject invention will preferablyproduce “an effective amount” of the protein so that when the plant istreated with a herbicide, the level of protein expression is sufficientto render the plant completely or partially resistant or tolerant to theherbicide (at a typical rate, unless otherwise specified; typicalapplication rates can be found in the well-known Herbicide Handbook(Weed Science Society of America, Eighth Edition, 2002), for example).The herbicide can be applied at rates that would normally kill thetarget plant, at normal field use rates and concentrations. (Because ofthe subject invention, the level and/or concentration can optionally behigher than those that were previously used.) Preferably, plant cellsand plants of the subject invention are protected against growthinhibition or injury caused by herbicide treatment. Transformed plantsand plant cells of the subject invention are preferably renderedresistant or tolerant to an herbicide, as discussed herein, meaning thatthe transformed plant and plant cells can grow in the presence ofeffective amounts of one or more herbicides as discussed herein.Preferred proteins of the subject invention have catalytic activity tometabolize one or more aryloxyalkanoate compounds.

One cannot easily discuss the term “resistance” and not use the verb“tolerate” or the adjective “tolerant.” The industry has spentinnumerable hours debating Herbicide Tolerant Crops (HTC) versusHerbicide Resistant Crops (HRC). ETC is a preferred term in theindustry. However, the official Weed Science Society of Americadefinition of resistance is “the inherited ability of a plant to surviveand reproduce following exposure to a dose of herbicide normally lethalto the wild type. In a plant, resistance may be naturally occurring orinduced by such techniques as genetic engineering or selection ofvariants produced by tissue culture or mutagenesis.” As used hereinunless otherwise indicated, herbicide “resistance” is heritable andallows a plant to grow and reproduce in the presence of a typicalherbicidally effective treatment by an herbicide for a given plant, assuggested by the current edition of The Herbicide Handbook as of thefiling of the subject disclosure. As is recognized by those skilled inthe art, a plant may still be considered “resistant” even though somedegree of plant injury from herbicidal exposure is apparent. As usedherein, the term “tolerance” is broader than the term “resistance,” andincludes “resistance” as defined herein, as well an improved capacity ofa particular plant to withstand the various degrees of herbicidallyinduced injury that typically result in wild-type plants of the samegenotype at the same herbicidal dose.

Transfer of the functional activity to plant or bacterial systems caninvolve a nucleic acid sequence, encoding the amino acid sequence for aprotein of the subject invention, integrated into a protein expressionvector appropriate to the host in which the vector will reside. One wayto obtain a nucleic acid sequence encoding a protein with functionalactivity is to isolate the native genetic material from the bacterialspecies which produce the protein of interest, using information deducedfrom the protein's amino acid sequence, as disclosed herein. The nativesequences can be optimized for expression in plants, for example, asdiscussed in more detail below. An optimized polynucleotide can also bedesigned based on the protein sequence.

The subject invention provides classes of proteins having novelactivities as identified herein. One way to characterize these classesof proteins and the polynucleotides that encode them is by defining apolynucleotide by its ability to hybridize, under a range of specifiedconditions, with an exemplified nucleotide sequence (the complementthereof and/or a probe or probes derived from either strand) and/or bytheir ability to be amplified by PCR using primers derived from theexemplified sequences.

There are a number of methods for obtaining proteins for use accordingto the subject invention. For example, antibodies to the proteinsdisclosed herein can be used to identify and isolate other proteins froma mixture of proteins. Specifically, antibodies may be raised to theportions of the proteins that are most conserved or most distinct, ascompared to other related proteins. These antibodies can then be used tospecifically identify equivalent proteins with the characteristicactivity by immunoprecipitation, enzyme linked immunosorbent assay(ELISA), or immuno-blotting. Antibodies to the proteins disclosedherein, or to equivalent proteins, or to fragments of these proteins,can be readily prepared using standard procedures. Such antibodies arean aspect of the subject invention. Antibodies of the subject inventioninclude monoclonal and polyclonal antibodies, preferably produced inresponse to an exemplified or suggested protein.

One skilled in the art would readily recognize that proteins (and genes)of the subject invention can be obtained from a variety of sources.Since entire herbicide degradation operons are known to be encoded ontransposable elements such as plasmids, as well as genomicallyintegrated, proteins of the subject invention can be obtained from awide variety of microorganisms, for example, including recombinantand/or wild-type bacteria.

Mutants of bacterial isolates can be made by procedures that are wellknown in the art. For example, asporogenous mutants can be obtainedthrough ethylmethane sulfonate (EMS) mutagenesis of an isolate. Themutant strains can also be made using ultraviolet light andnitrosoguanidine by procedures well known in the art.

A protein “from” or “obtainable from” any of the subject isolatesreferred to or suggested herein means that the protein (or a similarprotein) can be obtained from the isolate or some other source, such asanother bacterial strain or a plant. “Derived from” also has thisconnotation, and includes proteins obtainable from a given type ofbacterium that are modified for expression in a plant, for example. Oneskilled in the art will readily recognize that, given the disclosure ofa bacterial gene and protein, a plant can be engineered to produce theprotein. Antibody preparations, nucleic acid probes (DNA, RNA, or PNA,for example), and the like can be prepared using the polynucleotideand/or amino acid sequences disclosed herein and used to screen andrecover other related genes from other (natural) sources.

Standard molecular biology techniques may be used to clone and sequencethe proteins and genes described herein. Additional information may befound in Sambrook et al., 1989, which is incorporated herein byreference.

Polynucleotides and probes. The subject invention further providesnucleic acid sequences that encode proteins for use according to thesubject invention. The subject invention further provides methods ofidentifying and characterizing genes that encode proteins having thedesired herbicidal activity. In one embodiment, the subject inventionprovides unique nucleotide sequences that are useful as hybridizationprobes and/or primers for PCR techniques. The primers producecharacteristic gene fragments that can be used in the identification,characterization, and/or isolation of specific genes of interest. Thenucleotide sequences of the subject invention encode proteins that aredistinct from previously described proteins.

The polynucleotides of the subject invention can be used to formcomplete “genes” to encode proteins or peptides in a desired host cell.For example, as the skilled artisan would readily recognize, the subjectpolynucleotides can be appropriately placed under the control of apromoter in a host of interest, as is readily known in the art. Thelevel of gene expression and temporal/tissue specific expression cangreatly impact the utility of the invention. Generally, greater levelsof protein expression of a degradative gene will result in faster andmore complete degradation of a substrate (in this case a targetherbicide). Promoters will be desired to express the target gene at highlevels unless the high expression has a consequential negative impact onthe health of the plant. Typically, one would wish to have the AAD-13gene constitutively expressed in all tissues for complete protection ofthe plant at all growth stages. However, one could alternatively use avegetatively expressed resistance gene; this would allow use of thetarget herbicide in-crop for weed control and would subsequently controlsexual reproduction of the target crop by application during theflowering stage. In addition, desired levels and times of expression canalso depend on the type of plant and the level of tolerance desired.Some preferred embodiments use strong constitutive promoters combinedwith transcription enhancers and the like to increase expression levelsand to enhance tolerance to desired levels. Some such applications arediscussed in more detail below, before the Examples section.

As the skilled artisan knows, DNA typically exists in a double-strandedform. In this arrangement, one strand is complementary to the otherstrand and vice versa. As DNA is replicated in a plant (for example),additional complementary strands of DNA are produced. The “codingstrand” is often used in the art to refer to the strand that binds withthe anti-sense strand. The mRNA is transcribed from the “anti-sense”strand of DNA. The “sense” or “coding” strand has a series of codons (acodon is three nucleotides that can be read as a three-residue unit tospecify a particular amino acid) that can be read as an open readingframe (ORF) to form a protein or peptide of interest. In order toproduce a protein in vivo, a strand of DNA is typically transcribed intoa complementary strand of mRNA which is used as the template for theprotein. Thus, the subject invention includes the use of the exemplifiedpolynucleotides shown in the attached sequence listing and/orequivalents including the complementary strands. RNA and PNA (peptidenucleic acids) that are functionally equivalent to the exemplified DNAmolecules are included in the subject invention.

In one embodiment of the subject invention, bacterial isolates can becultivated under conditions resulting in high multiplication of themicrobe. After treating the microbe to provide single-stranded genomicnucleic acid, the DNA can be contacted with the primers of the inventionand subjected to PCR amplification. Characteristic fragments of genes ofinterest will be amplified by the procedure, thus identifying thepresence of the gene(s) of interest.

Further aspects of the subject invention include genes and isolatesidentified using the methods and nucleotide sequences disclosed herein.The genes thus identified can encode herbicidal resistance proteins ofthe subject invention.

Proteins and genes for use according to the subject invention can beidentified and obtained by using oligonucleotide probes, for example.These probes are detectable nucleotide sequences that can be detectableby virtue of an appropriate label or may be made inherently fluorescentas described in International Application No. WO 93/16094. The probes(and the polynucleotides of the subject invention) may be DNA, RNA, orPNA. In addition to adenine (A), cytosine (C), guanine (G), thymine (T),and uracil (U; for RNA molecules), synthetic probes (andpolynucleotides) of the subject invention can also have inosine (aneutral base capable of pairing with all four bases; sometimes used inplace of a mixture of all four bases in synthetic probes) and/or othersynthetic (non-natural) bases. Thus, where a synthetic, degenerateoligonucleotide is referred to herein, and “N” or “n” is usedgenerically, “N” or “n” can be G, A, T, C, or inosine. Ambiguity codesas used herein are in accordance with standard IUPAC naming conventionsas of the filing of the subject application (for example, R means A orG, Y means C or T, etc.).

As is well known in the art, if a probe molecule hybridizes with anucleic acid sample, it can be reasonably assumed that the probe andsample have substantial homology/similarity/identity. Preferably,hybridization of the polynucleotide is first conducted followed bywashes under conditions of low, moderate, or high stringency bytechniques well-known in the art, as described in, for example, Keller,G. H., M. M. Manak (1987) DNA Probes, Stockton Press, New York, N.Y.,pp. 169-170. For example, as stated therein, low stringency conditionscan be achieved by first washing with 2×SSC (Standard SalineCitrate)/0.1% SDS (Sodium Dodecyl Sulfate) for 15 minutes at roomtemperature. Two washes are typically performed. Higher stringency canthen be achieved by lowering the salt concentration and/or by raisingthe temperature. For example, the wash described above can be followedby two washings with 0.1×SSC/0.1% SDS for 15 minutes each at roomtemperature followed by subsequent washes with 0.1×SSC/0.1% SDS for 30minutes each at 55° C. These temperatures can be used with otherhybridization and wash protocols set forth herein and as would be knownto one skilled in the art (SSPE can be used as the salt instead of SSC,for example). The 2×SSC/0.1% SDS can be prepared by adding 50 ml of20×SSC and 5 ml of 10% SDS to 445 ml of water. 20×SSC can be prepared bycombining NaCl (175.3 g/0.150 M), sodium citrate (88.2 g/0.015 M), andwater, adjusting pH to 7.0 with 10 N NaOH, then adjusting the volume to1 liter. 10% SDS can be prepared by dissolving 10 g of SDS in 50 ml ofautoclaved water, then diluting to 100 ml.

Detection of the probe provides a means for determining in a knownmanner whether hybridization has been maintained. Such a probe analysisprovides a rapid method for identifying genes of the subject invention.The nucleotide segments used as probes according to the invention can besynthesized using a DNA synthesizer and standard procedures. Thesenucleotide sequences can also be used as PCR primers to amplify genes ofthe subject invention.

Hybridization characteristics of a molecule can be used to definepolynucleotides of the subject invention. Thus the subject inventionincludes polynucleotides (and/or their complements, preferably theirfull complements) that hybridize with a polynucleotide exemplifiedherein. That is, one way to define a gene (and the protein it encodes),for example, is by its ability to hybridize (under any of the conditionsspecifically disclosed herein) with a known or specifically exemplifiedgene.

As used herein, “stringent” conditions for hybridization refers toconditions which achieve the same, or about the same, degree ofspecificity of hybridization as the conditions employed by the currentapplicants. Specifically, hybridization of immobilized DNA on Southernblots with ³²P-labeled gene-specific probes can be performed by standardmethods (see, e.g., Maniatis et al. 1982). In general, hybridization andsubsequent washes can be carried out under conditions that allow fordetection of target sequences. For double-stranded DNA gene probes,hybridization can be carried out overnight at 20-25° C. below themelting temperature (Tm) of the DNA hybrid in 6×SSPE, 5×Denhardt'ssolution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature isdescribed by the following formula (Beltz et al. 1983):Tm=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.61(% formamide)−600/length ofduplex in base pairs.

Washes can typically be carried out as follows:

-   -   (1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS        (low stringency wash).    -   (2) Once at Tm-20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS        (moderate stringency wash).

For oligonucleotide probes, hybridization can be carried out overnightat 10-20° C. below the melting temperature (Tm) of the hybrid in 6×SSPE,5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm foroligonucleotide probes can be determined by the following formula:Tm(° C.)=2(number T/A base pairs)+4(number G/C base pairs)(Suggs et al., 1981).

Washes can typically be out as follows:

-   -   (1) Twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS        (low stringency wash).    -   (2) Once at the hybridization temperature for 15 minutes in        1×SSPE, 0.1% SDS (moderate stringency wash).

In general, salt and/or temperature can be altered to change stringency.With a labeled DNA fragment >70 or so bases in length, the followingconditions can be used:

-   -   Low: 1 or 2×SSPE, room temperature    -   Low: 1 or 2×SSPE, 42° C.    -   Moderate: 0.2× or 1×SSPE, 65° C.    -   High: 0.1×SSPE, 65° C.

Duplex formation and stability depend on substantial complementaritybetween the two strands of a hybrid, and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, the probe sequences ofthe subject invention include mutations (both single and multiple),deletions, insertions of the described sequences, and combinationsthereof, wherein said mutations, insertions and deletions permitformation of stable hybrids with the target polynucleotide of interest.Mutations, insertions, and deletions can be produced in a givenpolynucleotide sequence in many ways, and these methods are known to anordinarily skilled artisan. Other methods may become known in thefuture.

PCR technology. Polymerase Chain Reaction (PCR) is a repetitive,enzymatic, primed synthesis of a nucleic acid sequence. This procedureis well known and commonly used by those skilled in this art (seeMullis, U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki etal., 1985). PCR is based on the enzymatic amplification of a DNAfragment of interest that is flanked by two oligonucleotide primers thathybridize to opposite strands of the target sequence. The primers arepreferably oriented with the 3′ ends pointing towards each other.Repeated cycles of heat denaturation of the template, annealing of theprimers to their complementary sequences, and extension of the annealedprimers with a DNA polymerase result in the amplification of the segmentdefined by the 5′ ends of the PCR primers. The extension product of eachprimer can serve as a template for the other primer, so each cycleessentially doubles the amount of DNA fragment produced in the previouscycle. This results in the exponential accumulation of the specifictarget fragment, up to several million-fold in a few hours. By using athermostable DNA polymerase such as Taq polymerase, isolated from thethermophilic bacterium Thermus aquaticus, the amplification process canbe completely automated. Other enzymes which can be used are known tothose skilled in the art.

Exemplified DNA sequences, or segments thereof, can be used as primersfor PCR amplification. In performing PCR amplification, a certain degreeof mismatch can be tolerated between primer and template. Therefore,mutations, deletions, and insertions (especially additions ofnucleotides to the 5′ end) of the exemplified primers fall within thescope of the subject invention. Mutations, insertions, and deletions canbe produced in a given primer by methods known to an ordinarily skilledartisan.

Modification of genes and proteins. The subject genes and proteins canbe fused to other genes and proteins to produce chimeric or fusionproteins. The genes and proteins useful according to the subjectinvention include not only the specifically exemplified full-lengthsequences, but also portions, segments and/or fragments (includingcontiguous fragments and internal and/or terminal deletions compared tothe full-length molecules) of these sequences, variants, mutants,chimerics, and fusions thereof. Proteins of the subject invention canhave substituted amino acids so long as they retain desired functionalactivity. “Variant” genes have nucleotide sequences that encode the sameproteins or equivalent proteins having activity equivalent or similar toan exemplified protein.

The terms “variant proteins” and “equivalent proteins” refer to proteinshaving the same or essentially the same biological/functional activityagainst the target substrates and equivalent sequences as theexemplified proteins. As used herein, reference to an “equivalent”sequence refers to sequences having amino acid substitutions, deletions,additions, or insertions that improve or do not adversely affectactivity to a significant extent. Fragments retaining activity are alsoincluded in this definition. Fragments and other equivalents that retainthe same or similar function or activity as a corresponding fragment ofan exemplified protein are within the scope of the subject invention.Changes, such as amino acid substitutions or additions, can be made fora variety of purposes, such as increasing (or decreasing) proteasestability of the protein (without materially/substantially decreasingthe functional activity of the protein), removing or adding arestriction site, and the like. Variations of genes may be readilyconstructed using standard techniques for making point mutations, forexample.

In addition, U.S. Pat. No. 5,605,793, for example, describes methods forgenerating additional molecular diversity by using DNA reassembly afterrandom or focused fragmentation. This can be referred to as gene“shuffling,” which typically involves mixing fragments (of a desiredsize) of two or more different DNA molecules, followed by repeatedrounds of renaturation. This can improve the activity of a proteinencoded by a starting gene. The result is a chimeric protein havingimproved activity, altered substrate specificity, increased enzymestability, altered stereospecificity, or other characteristics.

“Shuffling” can be designed and targeted after obtaining and examiningthe atomic 3D (three dimensional) coordinates and crystal structure of aprotein of interest. Thus, “focused shuffling” can be directed tocertain segments of a protein that are ideal for modification, such assurface-exposed segments, and preferably not internal segments that areinvolved with protein folding and essential 3D structural integrity.

Specific changes to the “active site” of the enzyme can be made toaffect the inherent functionality with respect to activity orstereospecificity (see alignment FIG. 2) Muller et. al. (2006). Theknown tauD crystal structure was used as a model dioxygenase todetermine active site residues while bound to its inherent substratetaurine. Elkins et al. (2002) “X-ray crystal structure of Escerichiacoli taurine/alpha-ketoglutarate dioxygenase complexed to ferrous ironand substrates,” Biochemistry 41(16):5185-5192. Regarding sequenceoptimization and designability of enzyme active sites, see Chakrabartiet al., PNAS, (Aug. 23, 2005), 102(34):12035-12040.

Variant genes can be used to produce variant proteins; recombinant hostscan be used to produce the variant proteins. Using these “geneshuffling” techniques, equivalent genes and proteins can be constructedthat comprise any 5, 10, or 20 contiguous residues (amino acid ornucleotide) of any sequence exemplified herein. As one skilled in theart knows, the gene shuffling techniques, for example, can be adjustedto obtain equivalents having, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, or 288 contiguous residues (amino acid ornucleotide), corresponding to a segment (of the same size) in any of theexemplified or suggested sequences (or the complements (fullcomplements) thereof). Similarly sized segments, especially those forconserved regions, can also be used as probes and/or primers.

Fragments of full-length genes can be made using commercially availableexonucleases or endonucleases according to standard procedures. Forexample, enzymes such as Bal31 or site-directed mutagenesis can be usedto systematically cut off nucleotides from the ends of these genes.Also, genes that encode active fragments may be obtained using a varietyof restriction enzymes. Proteases may be used to directly obtain activefragments of these proteins.

It is within the scope of the invention as disclosed herein thatproteins can be truncated and still retain functional activity. By“truncated protein” it is meant that a portion of a protein may becleaved off while the remaining truncated protein retains and exhibitsthe desired activity after cleavage. Cleavage can be achieved by variousproteases. Furthermore, effectively cleaved proteins can be producedusing molecular biology techniques wherein the DNA bases encoding saidprotein are removed either through digestion with restrictionendonucleases or other techniques available to the skilled artisan.After truncation, said proteins can be expressed in heterologous systemssuch as E. coli, baculoviruses, plant-based viral systems, yeast, andthe like and then placed in insect assays as disclosed herein todetermine activity. It is well-known in the art that truncated proteinscan be successfully produced so that they retain functional activitywhile having less than the entire, full-length sequence. For example,B.t. proteins can be used in a truncated (core protein) form (see, e.g.,Höfte et al. (1989), and Adang et al. (1985)). As used herein, the term“protein” can include functionally active truncations.

In some cases, especially for expression in plants, it can beadvantageous to use truncated genes that express truncated proteins.Preferred truncated genes will typically encode 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or99% of the full-length protein.

Certain proteins of the subject invention have been specificallyexemplified herein. As these proteins are merely exemplary of theproteins of the subject invention, it should be readily apparent thatthe subject invention comprises variant or equivalent proteins (andnucleotide sequences coding for equivalents thereof) having the same orsimilar activity of the exemplified proteins. Equivalent proteins willhave amino acid similarity (and/or homology) with an exemplifiedprotein. The amino acid identity will typically be at least 60%,preferably at least 75%, more preferably at least 80%, even morepreferably at least 90%, and can be at least 95%. Preferred proteins ofthe subject invention can also be defined in terms of more particularidentity and/or similarity ranges. For example, the identity and/orsimilarity can be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, or 99% as compared to a sequence exemplified or suggested herein.Any number listed above can be used to define the upper and lowerlimits.

Unless otherwise specified, as used herein, percent sequence identityand/or similarity of two nucleic acids is determined using the algorithmof Karlin and Altschul, 1990, modified as in Karlin and Altschul 1993.Such an algorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al., 1990. BLAST nucleotide searches are performed with theNBLAST program, score=100, wordlength=12. Gapped BLAST can be used asdescribed in Altschul et al., 1997. When utilizing BLAST and GappedBLAST programs, the default parameters of the respective programs(NBLAST and XBLAST) are used. See NCBI/NIH website. To obtain gappedalignments for comparison purposes, the AlignX function of Vector NTISuite 8 (InforMax, Inc., North Bethesda, Md., U.S.A.), was usedemploying the default parameters. These were: a Gap opening penalty of15, a Gap extension penalty of 6.66, and a Gap separation penalty rangeof 8.

Various properties and three-dimensional features of the protein canalso be changed without adversely affecting the activity/functionalityof the protein. Conservative amino acid substitutions can betolerated/made to not adversely affect the activity and/orthree-dimensional configuration of the molecule. Amino acids can beplaced in the following classes: non-polar, uncharged polar, basic, andacidic. Conservative substitutions whereby an amino acid of one class isreplaced with another amino acid of the same type fall within the scopeof the subject invention so long as the substitution is not adverse tothe biological activity of the compound. Table 2 provides a listing ofexamples of amino acids belonging to each class.

TABLE 2 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made.However, preferred substitutions do not significantly detract from thefunctional/biological activity of the protein.

As used herein, reference to “isolated” polynucleotides and/or“purified” proteins refers to these molecules when they are notassociated with the other molecules with which they would be found innature. Thus, reference to “isolated” and/or “purified” signifies theinvolvement of the “hand of man” as described herein. For example, abacterial “gene” of the subject invention put into a plant forexpression is an “isolated polynucleotide.” Likewise, a protein derivedfrom a bacterial protein and produced by a plant is an “isolatedprotein.”

Because of the degeneracy/redundancy of the genetic code, a variety ofdifferent DNA sequences can encode the amino acid sequences disclosedherein. It is well within the skill of a person trained in the art tocreate alternative DNA sequences that encode the same, or essentiallythe same, proteins. These variant DNA sequences are within the scope ofthe subject invention. This is also discussed in more detail below inthe section entitled “Optimization of sequence for expression inplants.”

Optimization of sequence for expression in plants. To obtain highexpression of heterologous genes in plants it is generally preferred toreengineer the genes so that they are more efficiently expressed in (thecytoplasm of) plant cells. Maize is one such plant where it may bepreferred to re-design the heterologous gene(s) prior to transformationto increase the expression level thereof in said plant. Therefore, anadditional step in the design of genes encoding a bacterial protein isreengineering of a heterologous gene for optimal expression, using codonbias more closely aligned with the target plant sequence, whether adicot or monocot species. Sequences can also be optimized for expressionin any of the more particular types of plants discussed elsewhereherein.

Transgenic hosts. The protein-encoding genes of the subject inventioncan be introduced into a wide variety of microbial or plant hosts. Thesubject invention includes transgenic plant cells and transgenic plants.Preferred plants (and plant cells) are corn, Arabidopsis, tobacco,soybeans, cotton, canola, rice, wheat, turf, legume forages (e.g.,alfalfa and clover), pasture grasses, and the like. Other types oftransgenic plants can also be made according to the subject invention,such as fruits, vegetables, ornamental plants, and trees. Moregenerally, dicots and/or monocots can be used in various aspects of thesubject invention.

In preferred embodiments, expression of the gene results, directly orindirectly, in the intracellular production (and maintenance) of theprotein(s) of interest. Plants can be rendered herbicide-resistant inthis manner. Such hosts can be referred to as transgenic, recombinant,transformed, and/or transfected hosts and/or cells. In some aspects ofthis invention (when cloning and preparing the gene of interest, forexample), microbial (preferably bacterial) cells can be produced andused according to standard techniques, with the benefit of the subjectdisclosure.

Plant cells transfected with a polynucleotide of the subject inventioncan be regenerated into whole plants. The subject invention includescell cultures including tissue cell cultures, liquid cultures, andplated cultures. Seeds produced by and/or used to generate plants of thesubject invention are also included within the scope of the subjectinvention. Other plant tissues and parts are also included in thesubject invention. The subject invention likewise includes methods ofproducing plants or cells comprising a polynucleotide of the subjectinvention. One preferred method of producing such plants is by plantinga seed of the subject invention.

Although plants are preferred, the subject invention also includesproduction of highly active recombinant AAD-13 in a Pseudomonasfluorescens (Pf) host strain, for example. The subject inventionincludes preferred growth temperatures for maintaining soluble activeAAD-13 in this host and a formulation process that can store and restoreAAD-13 activity in solution; and a lyophilization process that canretain AAD-13 activity for long-term storage and shelf life.

Insertion of genes to form transgenic hosts. One aspect of the subjectinvention is the transformation/transfection of plants, plant cells, andother host cells with polynucleotides of the subject invention thatexpress proteins of the subject invention. Plants transformed in thismanner can be rendered resistant to a variety of herbicides withdifferent modes of action.

A wide variety of methods are available for introducing a gene encodinga desired protein into the target host under conditions that allow forstable maintenance and expression of the gene. These methods are wellknown to those skilled in the art and are described, for example, inU.S. Pat. No. 5,135,867.

Vectors comprising an AAD-13 polynucleotide are included in the scope ofthe subject invention. For example, a large number of cloning vectorscomprising a replication system in E. coli and a marker that permitsselection of the transformed cells are available for preparation for theinsertion of foreign genes into higher plants. The vectors comprise, forexample, pBR322, pUC series, M13 mp series, pACYC184, etc. Accordingly,the sequence encoding the protein can be inserted into the vector at asuitable restriction site. The resulting plasmid is used fortransformation into E. coli. The E. coli cells are cultivated in asuitable nutrient medium, then harvested and lysed. The plasmid isrecovered by purification away from genomic DNA. Sequence analysis,restriction analysis, electrophoresis, and other biochemical-molecularbiological methods are generally carried out as methods of analysis.After each manipulation, the DNA sequence used can be restrictiondigested and joined to the next DNA sequence. Each plasmid sequence canbe cloned in the same or other plasmids. Depending on the method ofinserting desired genes into the plant, other DNA sequences may benecessary. If, for example, the Ti or Ri plasmid is used for thetransformation of the plant cell, then at least the right border, butoften the right and the left border of the Ti or Ri plasmid T-DNA, hasto be joined as the flanking region of the genes to be inserted. The useof T-DNA for the transformation of plant cells has been intensivelyresearched and described in EP 120 516; Hoekema (1985); Fraley et al.(1986); and An et al. (1985).

A large number of techniques are available for inserting DNA into aplant host cell. Those techniques include transformation with T-DNAusing Agrobacterium tumefaciens or Agrobacterium rhizogenes astransformation agent, fusion, injection, biolistics (microparticlebombardment), silicon carbide whiskers, aerosol beaming, PEG, orelectroporation as well as other possible methods. If Agrobacteria areused for the transformation, the DNA to be inserted has to be clonedinto special plasmids, namely either into an intermediate vector or intoa binary vector. The intermediate vectors can be integrated into the Tior Ri plasmid by homologous recombination owing to sequences that arehomologous to sequences in the T-DNA. The Ti or Ri plasmid alsocomprises the vir region necessary for the transfer of the T-DNA.Intermediate vectors cannot replicate themselves in Agrobacteria. Theintermediate vector can be transferred into Agrobacterium tumefaciens bymeans of a helper plasmid (conjugation). Binary vectors can replicatethemselves both in E. coli and in Agrobacteria. They comprise aselection marker gene and a linker or polylinker which are framed by theright and left T-DNA border regions. They can be transformed directlyinto Agrobacteria (Holsters, 1978). The Agrobacterium used as host cellis to comprise a plasmid carrying a vir region. The vir region isnecessary for the transfer of the T-DNA into the plant cell. AdditionalT-DNA may be contained. The bacterium so transformed is used for thetransformation of plant cells. Plant explants can be cultivatedadvantageously with Agrobacterium tumefaciens or Agrobacteriumrhizogenes for the transfer of the DNA into the plant cell. Whole plantscan then be regenerated from the infected plant material (for example,pieces of leaf, segments of stalk, roots, but also protoplasts orsuspension-cultivated cells) in a suitable medium, which may containantibiotics or biocides for selection. The plants so obtained can thenbe tested for the presence of the inserted DNA. No special demands aremade of the plasmids in the case of injection and electroporation. It ispossible to use ordinary plasmids, such as, for example, pUCderivatives.

The transformed cells grow inside the plants in the usual manner. Theycan form germ cells and transmit the transformed trait(s) to progenyplants. Such plants can be grown in the normal manner and crossed withplants that have the same transformed hereditary factors or otherhereditary factors. The resulting hybrid individuals have thecorresponding phenotypic properties.

In some preferred embodiments of the invention, genes encoding thebacterial protein are expressed from transcriptional units inserted intothe plant genome. Preferably, said transcriptional units are recombinantvectors capable of stable integration into the plant genome and enableselection of transformed plant lines expressing mRNA encoding theproteins.

Once the inserted DNA has been integrated in the genome, it isrelatively stable there (and does not come out again). It normallycontains a selection marker that confers on the transformed plant cellsresistance to a biocide or an antibiotic, such as kanamycin, G418,bleomycin, hygromycin, or chloramphenicol, inter alia. Plant selectablemarkers also typically can provide resistance to various herbicides suchas glufosinate (e.g., PAT/bar), glyphosate (EPSPS), ALS-inhibitors(e.g., imidazolinone, sulfonylurea, triazolopyrimidine sulfonanilide, etal.), bromoxynil, HPPD-inhibitor resistance, PP O-inhibitors, ACC-aseinhibitors, and many others. The individually employed marker shouldaccordingly permit the selection of transformed cells rather than cellsthat do not contain the inserted DNA. The gene(s) of interest arepreferably expressed either by constitutive or inducible promoters inthe plant cell. Once expressed, the mRNA is translated into proteins,thereby incorporating amino acids of interest into protein. The genesencoding a protein expressed in the plant cells can be under the controlof a constitutive promoter, a tissue-specific promoter, or an induciblepromoter.

Several techniques exist for introducing foreign recombinant vectorsinto plant cells, and for obtaining plants that stably maintain andexpress the introduced gene. Such techniques include the introduction ofgenetic material coated onto microparticles directly into cells (U.S.Pat. Nos. 4,945,050 to Cornell and 5,141,131 to DowElanco, now DowAgroSciences, LLC). In addition, plants may be transformed usingAgrobacterium technology, see U.S. Pat. Nos. 5,177,010 to University ofToledo; 5,104,310 to Texas A&M; European Patent Application 0131624B1;European Patent Applications 120516, 159418B1 and 176,112 toSchilperoot; U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763 and4,940,838 and 4,693,976 to Schilperoot; European Patent Applications116718, 290799, 320500, all to Max Planck; European Patent Applications604662 and 627752, and U.S. Pat. No. 5,591,616, to Japan Tobacco;European Patent Applications 0267159 and 0292435, and U.S. Pat. No.5,231,019, all to Ciba Geigy, now Syngenta; U.S. Pat. Nos. 5,463,174 and4,762,785, both to Calgene; and U.S. Pat. Nos. 5,004,863 and 5,159,135,both to Agracetus. Other transformation technology includes whiskerstechnology. See U.S. Pat. Nos. 5,302,523 and 5,464,765, both to Zeneca,now Syngenta. Other direct DNA delivery transformation technologyincludes aerosol beam technology. See U.S. Pat. No. 6,809,232.Electroporation technology has also been used to transform plants. SeeWO 87/06614 to Boyce Thompson Institute; U.S. Pat. Nos. 5,472,869 and5,384,253, both to Dekalb; and WO 92/09696 and WO 93/21335, both toPlant Genetic Systems. Furthermore, viral vectors can also be used toproduce transgenic plants expressing the protein of interest. Forexample, monocotyledonous plants can be transformed with a viral vectorusing the methods described in U.S. Pat. No. 5,569,597 to Mycogen PlantScience and Ciba-Geigy (now Syngenta), as well as U.S. Pat. Nos.5,589,367 and 5,316,931, both to Biosource, now Large Scale Biology.

As mentioned previously, the manner in which the DNA construct isintroduced into the plant host is not critical to this invention. Anymethod that provides for efficient transformation may be employed. Forexample, various methods for plant cell transformation are describedherein and include the use of Ti or Ri-plasmids and the like to performAgrobacterium mediated transformation. In many instances, it will bedesirable to have the construct used for transformation bordered on oneor both sides by T-DNA borders, more specifically the right border. Thisis particularly useful when the construct uses Agrobacterium tumefaciensor Agrobacterium rhizogenes as a mode for transformation, although T-DNAborders may find use with other modes of transformation. WhereAgrobacterium is used for plant cell transformation, a vector may beused which may be introduced into the host for homologous recombinationwith T-DNA or the Ti or Ri plasmid present in the host. Introduction ofthe vector may be performed via electroporation, tri-parental mating andother techniques for transforming gram-negative bacteria which are knownto those skilled in the art. The manner of vector transformation intothe Agrobacterium host is not critical to this invention. The Ti or Riplasmid containing the T-DNA for recombination may be capable orincapable of causing gall formation, and is not critical to saidinvention so long as the vir genes are present in said host.

In some cases where Agrobacterium is used for transformation, theexpression construct being within the T-DNA borders will be insertedinto a broad spectrum vector such as pRK2 or derivatives thereof asdescribed in Ditta et al. (1980) and EPO 0 120 515. Included within theexpression construct and the T-DNA will be one or more markers asdescribed herein which allow for selection of transformed Agrobacteriumand transformed plant cells. The particular marker employed is notessential to this invention, with the preferred marker depending on thehost and construction used.

For transformation of plant cells using Agrobacterium, explants may becombined and incubated with the transformed Agrobacterium for sufficienttime to allow transformation thereof. After transformation, theAgrobacteria are killed by selection with the appropriate antibiotic andplant cells are cultured with the appropriate selective medium. Oncecalli are formed, shoot formation can be encouraged by employing theappropriate plant hormones according to methods well known in the art ofplant tissue culturing and plant regeneration. However, a callusintermediate stage is not always necessary. After shoot formation, saidplant cells can be transferred to medium which encourages root formationthereby completing plant regeneration. The plants may then be grown toseed and said seed can be used to establish future generations.Regardless of transformation technique, the gene encoding a bacterialprotein is preferably incorporated into a gene transfer vector adaptedto express said gene in a plant cell by including in the vector a plantpromoter regulatory element, as well as 3′ non-translatedtranscriptional termination regions such as Nos and the like.

In addition to numerous technologies for transforming plants, the typeof tissue that is contacted with the foreign genes may vary as well.Such tissue would include but would not be limited to embryogenictissue, callus tissue types I, II, and III, hypocotyl, meristem, roottissue, tissues for expression in phloem, and the like. Almost all planttissues may be transformed during dedifferentiation using appropriatetechniques described herein.

As mentioned above, a variety of selectable markers can be used, ifdesired. Preference for a particular marker is at the discretion of theartisan, but any of the following selectable markers may be used alongwith any other gene not listed herein which could function as aselectable marker. Such selectable markers include but are not limitedto aminoglycoside phosphotransferase gene of transposon Tn5 (Aph II)which encodes resistance to the antibiotics kanamycin, neomycin and G41;hygromycin resistance; methotrexate resistance, as well as those geneswhich encode for resistance or tolerance to glyphosate; phosphinothricin(bialaphos or glufosinate); ALS-inhibiting herbicides (imidazolinones,sulfonylureas and triazolopyrimidine herbicides), ACC-ase inhibitors(e.g., ayryloxypropionates or cyclohexanediones), and others such asbromoxynil, and HPPD-inhibitors (e.g., mesotrione) and the like.

In addition to a selectable marker, it may be desirous to use a reportergene. In some instances a reporter gene may be used with or without aselectable marker. Reporter genes are genes that are typically notpresent in the recipient organism or tissue and typically encode forproteins resulting in some phenotypic change or enzymatic property.Examples of such genes are provided in Weising et al., 1988. Preferredreporter genes include the beta-glucuronidase (GUS) of the uidA locus ofE. coli, the chloramphenicol acetyl transferase gene from Tn9 of E.coli, the green fluorescent protein from the bioluminescent jellyfishAequorea victoria, and the luciferase genes from firefly Photinuspyralis. An assay for detecting reporter gene expression may then beperformed at a suitable time after said gene has been introduced intorecipient cells. A preferred such assay entails the use of the geneencoding beta-glucuronidase (GUS) of the uidA locus of E. coli asdescribed by Jefferson et al., (1987) to identify transformed cells.

In addition to plant promoter regulatory elements, promoter regulatoryelements from a variety of sources can be used efficiently in plantcells to express foreign genes. For example, promoter regulatoryelements of bacterial origin, such as the octopine synthase promoter,the nopaline synthase promoter, the mannopine synthase promoter;promoters of viral origin, such as the cauliflower mosaic virus (35S and19S), 35T (which is a re-engineered 35S promoter, see U.S. Pat. No.6,166,302, especially Example 7E) and the like may be used. Plantpromoter regulatory elements include but are not limited toribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu),beta-conglycinin promoter, beta-phaseolin promoter, ADH promoter,heat-shock promoters, and tissue specific promoters. Other elements suchas matrix attachment regions, scaffold attachment regions, introns,enhancers, polyadenylation sequences and the like may be present andthus may improve the transcription efficiency or DNA integration. Suchelements may or may not be necessary for DNA function, although they canprovide better expression or functioning of the DNA by affectingtranscription, mRNA stability, and the like. Such elements may beincluded in the DNA as desired to obtain optimal performance of thetransformed DNA in the plant. Typical elements include but are notlimited to Adh-intron 1, Adh-intron 6, the alfalfa mosaic virus coatprotein leader sequence, osmotin UTR sequences, the maize streak viruscoat protein leader sequence, as well as others available to a skilledartisan. Constitutive promoter regulatory elements may also be usedthereby directing continuous gene expression in all cells types and atall times (e.g., actin, ubiquitin, CaMV 35S, and the like). Tissuespecific promoter regulatory elements are responsible for geneexpression in specific cell or tissue types, such as the leaves or seeds(e.g., zein, oleosin, napin, ACP, globulin and the like) and these mayalso be used.

Promoter regulatory elements may also be active (or inactive) during acertain stage of the plant's development as well as active in planttissues and organs. Examples of such include but are not limited topollen-specific, embryo-specific, corn-silk-specific,cotton-fiber-specific, root-specific, seed-endosperm-specific, orvegetative phase-specific promoter regulatory elements and the like.Under certain circumstances it may be desirable to use an induciblepromoter regulatory element, which is responsible for expression ofgenes in response to a specific signal, such as: physical stimulus (heatshock genes), light (RUBP carboxylase), hormone (Em), metabolites,chemical (tetracycline responsive), and stress. Other desirabletranscription and translation elements that function in plants may beused. Numerous plant-specific gene transfer vectors are known in theart.

Plant RNA viral based systems can also be used to express bacterialprotein. In so doing, the gene encoding a protein can be inserted intothe coat promoter region of a suitable plant virus which will infect thehost plant of interest. The protein can then be expressed thus providingprotection of the plant from herbicide damage. Plant RNA viral basedsystems are described in U.S. Pat. No. 5,500,360 to Mycogen PlantSciences, Inc. and U.S. Pat. Nos. 5,316,931 and 5,589,367 to Biosource,now Large Scale Biology.

Means of further increasing tolerance or resistance levels. It is shownherein that plants of the subject invention can be imparted with novelherbicide resistance traits without observable adverse effects onphenotype including yield. Such plants are within the scope of thesubject invention. Plants exemplified and suggested herein can withstand2×, 3×, 4×, and 5× typical application levels, for example, of at leastone subject herbicide. Improvements in these tolerance levels are withinthe scope of this invention. For example, various techniques are know inthe art, and can foreseeably be optimized and further developed, forincreasing expression of a given gene.

One such method includes increasing the copy number of the subjectAAD-13 genes (in expression cassettes and the like). Transformationevents can also be selected for those having multiple copies of thegenes.

Strong promoters and enhancers can be used to “supercharge” expression.Examples of such promoters include the preferred 35T promoter which uses35S enhancers. 35S, maize ubiquitin, Arabidopsis ubiquitin, A.t. actin,and CSMV promoters are included for such uses. Other strong viralpromoters are also preferred. Enhancers include 4 OCS and the 35S doubleenhancer. Matrix attachment regions (MARs) can also be used to increasetransformation efficiencies and transgene expression, for example.

Shuffling (directed evolution) and transcription factors can also beused for embodiments according to the subject invention.

Variant proteins can also be designed that differ at the sequence levelbut that retain the same or similar overall essential three-dimensionalstructure, surface charge distribution, and the like. See e.g. U.S. Pat.No. 7,058,515; Larson et al., Protein Sci. 2002 11: 2804-2813,“Thoroughly sampling sequence space: Large-scale protein design ofstructural ensembles.”; Crameri et al., Nature Biotechnology 15, 436-438(1997), “Molecular evolution of an arsenate detoxification pathway byDNA shuffling.”; Stemmer, W. P. C. 1994. DNA shuffling by randomfragmentation and reassembly: in vitro recombination for molecularevolution. Proc. Natl. Acad. Sci. USA 91: 10747-10751; Stemmer, W. P. C.1994. Rapid evolution of a protein in vitro by DNA shuffling. Nature370: 389-391; Stemmer, W. P. C. 1995. Searching sequence space.Bio/Technology 13: 549-553; Crameri, A., Cwirla, S. and Stemmer, W. P.C. 1996. Construction and evolution of antibody-phage libraries by DNAshuffling. Nature Medicine 2: 100-103; and Crameri, A., Whitehorn, E.A., Tate, E. and Stemmer, W. P. C. 1996. Improved green fluorescentprotein by molecular evolution using DNA shuffling. Nature Biotechnology14: 315-319.

The activity of recombinant polynucleotides inserted into plant cellscan be dependent upon the influence of endogenous plant DNA adjacent theinsert. Thus, another option is taking advantage of events that areknown to be excellent locations in a plant genome for insertions. Seee.g. WO 2005/103266 A1, relating to cry1F and cry1Ac cotton events; thesubject AAD-13 gene can be substituted in those genomic loci in place ofthe cry1F and/or cry1Ac inserts. Thus, targeted homologousrecombination, for example, can be used according to the subjectinvention. This type of technology is the subject of, for example, WO03/080809 A2 and the corresponding published U.S. application (USPA20030232410), relating to the use of zinc fingers for targetedrecombination. The use of recombinases (cre-lox and flp-frt for example)is also known in the art.

AAD-13 detoxification is believed to occur in the cytoplasm. Thus, meansfor further stabilizing this protein and mRNAs (including blocking mRNAdegradation) are included in aspects of the subject invention, andart-known techniques can be applied accordingly. The subject proteinscan be designed to resist degradation by proteases and the like(protease cleavage sites can be effectively removed by re-engineeringthe amino acid sequence of the protein). Such embodiments include theuse of 5′ and 3′ stem loop structures like UTRs from osmotin, and per5(AU-rich untranslated 5′ sequences). 5′ caps like 7-methyl or2′-O-methyl groups, e.g., 7-methylguanylic acid residue, can also beused. See, e.g.: Proc. Natl. Acad. Sci. USA Vol. 74, No. 7, pp.2734-2738 (July 1977) Importance of 5′-terminal blocking structure tostabilize mRNA in eukaryotic protein synthesis. Protein complexes orligand blocking groups can also be used.

Computational design of 5′ or 3′ UTR most suitable for AAD-13 (synthetichairpins) can also be conducted within the scope of the subjectinvention. Computer modeling in general, as well as gene shuffling anddirected evolution, are discussed elsewhere herein. More specificallyregarding computer modeling and UTRs, computer modeling techniques foruse in predicting/evaluating 5′ and 3′ UTR derivatives of the presentinvention include, but are not limited to: MFold version 3.1 availablefrom Genetics Corporation Group, Madison, Wis. (see Zucker et al.,Algorithms and Thermodynamics for RNA Secondary Structure Prediction: APractical Guide. In RNA Biochemistry and Biotechnology, 11-43, J.Barciszewski & B. F. C. Clark, eds., NATO ASI Series, Kluwer AcademicPublishers, Dordrecht, N L, (1999); Zucker et al., Expanded SequenceDependence of Thermodynamic Parameters Improves Prediction of RNASecondary Structure. J. Mol. Biol. 288, 911-940 (1999); Zucker et al.,RNA Secondary Structure Prediction. In Current Protocols in Nucleic AcidChemistry S. Beaucage, D. E. Bergstrom, G. D. Glick, and R. A. Joneseds., John Wiley & Sons, New York, 11.2.1-11.2.10, (2000)), COVE (RNAstructure analysis using covariance models (stochastic context freegrammar methods)) v.2.4.2 (Eddy & Durbin, Nucl. Acids Res. 1994, 22:2079-2088) which is freely distributed as source code and which can bedownloaded by accessing the website genetics.wustl.edu/eddy/software/,and FOLDALIGN, also freely distributed and available for downloading atthe website bioinf.au.dk. FOLDALIGN/ (see Finding the most significantcommon sequence and structure motifs in a set of RNA sequences. J.Gorodkin, L. J. Heyer and G. D. Stormo. Nucleic Acids Research, Vol. 25,no. 18 pp 3724-3732, 1997; Finding Common Sequence and Structure Motifsin a set of RNA Sequences. J. Gorodkin, L. J. Heyer, and G. D. Stormo.ISMB 5; 120-123, 1997).

Embodiments of the subject invention can be used in conjunction withnaturally evolved or chemically induced mutants (mutants can be selectedby screening techniques, then transformed with AAD-13 and possibly othergenes). Plants of the subject invention can be combined with ALSresistance and/or evolved glyphosate resistance. Aminopyralidresistance, for example, can also be combined or “stacked” with anAAD-13 gene.

Traditional breeding techniques can also be combined with the subjectinvention to powerfully combine, introgress, and improve desired traits.

Further improvements also include use with appropriate safeners tofurther protect plants and/or to add cross resistance to moreherbicides. (Safeners typically act to increase plants immune system byactivating/expressing cP450. Safeners are chemical agents that reducethe phytotoxicity of herbicides to crop plants by a physiological ormolecular mechanism, without compromising weed control efficacy.)

Herbicide safeners include benoxacor, cloquintocet, cyometrinil,dichlormid, dicyclonon, dietholate, fenchlorazole, fenclorim, flurazole,fluxofenim, furilazole, isoxadifen, mefenpyr, mephenate, naphthalicanhydride, and oxabetrinil. Plant activators (a new class of compoundsthat protect plants by activating their defense mechanisms) can also beused in embodiments of the subject invention. These include acibenzolarand probenazole.

Commercialized safeners can be used for the protection of large-seededgrass crops, such as corn, grain sorghum, and wet-sown rice, againstpreplant-incorporated or preemergence-applied herbicides of thethiocarbamate and chloroacetanilide families. Safeners also have beendeveloped to protect winter cereal crops such as wheat againstpostemergence applications of aryloxyphenoxypropionate and sulfonylureaherbicides. The use of safeners for the protection of corn and riceagainst sulfonylurea, imidazolinone, cyclohexanedione, isoxazole, andtriketone herbicides is also well-established. A safener-inducedenhancement of herbicide detoxification in safened plants is widelyaccepted as the major mechanism involved in safener action. Safenersinduce cofactors such as glutathione and herbicide-detoxifying enzymessuch as glutathione S-transferases, cytochrome P450 monooxygenases, andglucosyl transferases. Hatzios K K, Burgos N (2004) “Metabolism-basedherbicide resistance: regulation by safeners,” Weed Science: Vol. 52,No. 3 pp. 454-467.

Use of a cytochrome p4-50 monooxygenase gene stacked with AAD-13 is onepreferred embodiment. There are P450s involved in herbicide metabolism;cP450 can be of mammalian or plant origin, for example. In higherplants, cytochrome P450 monooxygenase (P450) is known to conductsecondary metabolism. It also plays an important role in the oxidativemetabolism of xenobiotics in cooperation with NADPH-cytochrome P450oxidoreductase (reductase). Resistance to some herbicides has beenreported as a result of the metabolism by P450 as well as glutathioneS-transferase. A number of microsomal P450 species involved inxenobiotic metabolism in mammals have been characterized by molecularcloning. Some of them were reported to metabolize several herbicidesefficiently. Thus, transgenic plants with plant or mammalian P450 canshow resistance to several herbicides.

One preferred embodiment of the foregoing is the use cP450 forresistance to acetochlor (acetochlor-based products include Surpass®,Keystone®, Keystone LA, FulTime® and TopNotch® herbicides) and/ortrifluralin (such as Treflan®). Such resistance in soybeans and/or cornis included in some preferred embodiments. For additional guidanceregarding such embodiments, see e.g. Inui et al., “A selectable markerusing cytochrome P450 monooxygenases for Arabidopsis transformation,”Plant Biotechnology 22, 281-286 (2005) (relating to a selection systemfor transformation of Arabidopsis thaliana via Agrobacterium tumefaciensthat uses human cytochrome P450 monooxygenases that metabolizeherbicides; herbicide tolerant seedlings were transformed and selectedwith the herbicides acetochlor, amiprophos-methyl, chlorpropham,chlorsulfuron, norflurazon, and pendimethalin); Siminszky et al.,“Expression of a soybean cytochrome P450 monooxygenase cDNA in yeast andtobacco enhances the metabolism of phenylurea herbicides,” PNAS Vol. 96,Issue 4, 1750-1755, Feb. 16, 1999; Sheldon et al, Weed Science: Vol. 48,No. 3, pp. 291-295, “A cytochrome P450 monooxygenase cDNA (CYP71A 10)confers resistance to linuron in transgenic Nicotiana tabacum”; and“Phytoremediation of the herbicides atrazine and metolachlor bytransgenic rice plants expressing human CYP1A1, CYP2B6, and CYP2C19,” JAgric Food Chem. 2006 Apr. 19; 54(8):2985-91 (relating to testing ahuman cytochrome p450 monooxygenase in rice where the rice plantsreportedly showed high tolerance to chloroacetomides (acetochlor,alachlor, metoachlor, pretilachlor, and thenylchlor), oxyacetamides(mefenacet), pyridazinones (norflurazon), 2,6-dinitroanalines(trifluralin and pendimethalin), phosphamidates (amiprofos-methyl,thiocarbamates (pyributicarb), and ureas (chlortoluron)).

There is also the possibility of altering or using different 2,4-Dchemistries to make the subject AAD-13 gene more efficient. Suchpossible changes include creating better substrates and better leavinggroups (higher electronegativity).

Auxin transport inhibitors (e.g. diflufcnzopyr) can also be used toincrease herbicide activity with 2,4-D.

Unless specifically indicated or implied, the terms “a”, “an”, and “the”signify “at least one” as used herein.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

Following are examples that illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

Example 1 Method for Identifying Genes that Impart Herbicide Resistancein Planta

As a way to identify genes which possess herbicide degrading activitiesin planta, it is possible to mine current public databases such as NCBI(National Center for Biotechnology Information). To begin the process,it is necessary to have a functional gene sequence already identifiedthat encodes a protein with the desired characteristics (i.e.,α-ketoglutarate dioxygenase activity). This protein sequence is thenused as the input for the BLAST (Basic Local Alignment Search Tool)(Altschul et al., 1997) algorithm to compare against available NCBIprotein sequences deposited. Using default settings, this search returnsupwards of 100 homologous protein sequences at varying levels. Theserange from highly identical (85-98%) to very low identity (23-35%) atthe amino acid level. Traditionally only sequences with high homologywould be expected to retain similar properties to the input sequence. Inthis case, only sequences with ≦50% homology were chosen. As exemplifiedherein, cloning and recombinantly expressing homologues with as littleas 35% amino acid conservation (relative to tfdA from Ralstoniaeutropha) can be used to impart commercial levels of resistance not onlyto the intended herbicide, but also to substrates never previouslytested with these enzymes.

A single gene (sdpA) was identified from the NCBI database (see thencbi.nlm.nih.gov website; accession #AJ628860) as a homologue with only35% amino acid identity to tfdA. Percent identity was determined byfirst translating both the sdpA and tfdA DNA sequences deposited in thedatabase to proteins, then using ClustalW in the VectorNTI softwarepackage to perform the multiple sequence alignment.

Example 2 Optimization of Sequence for Expression in Plants and Bacteria

2.1—Background.

To obtain higher levels of expression of heterologous genes in plants,it may be preferred to re-engineer the protein encoding sequence of thegenes so that they are more efficiently expressed in plant cells. Maizeis one such plant where it may be preferred to re-design theheterologous protein coding region prior to transformation to increasethe expression level of the gene and the level of encoded protein in theplant. Therefore, an additional step in the design of genes encoding abacterial protein is re-engineering of a heterologous gene for optimalexpression.

One reason for the re-engineering of a bacterial gene for expression inmaize is due to the non-optimal G+C content of the native gene. Forexample, the very low G+C content of many native bacterial gene(s) (andconsequent skewing towards high A+T content) results in the generationof sequences mimicking or duplicating plant gene control sequences thatare known to be highly A+T rich. The presence of some A+T-rich sequenceswithin the DNA of gene(s) introduced into plants (e.g., TATA box regionsnormally found in gene promoters) may result in aberrant transcriptionof the gene(s). On the other hand, the presence of other regulatorysequences residing in the transcribed mRNA (e.g., polyadenylation signalsequences such as AAUAAA, or sequences complementary to small nuclearRNAs involved in pre-mRNA splicing) may lead to RNA instability.Therefore, one goal in the design of genes encoding a bacterial proteinfor maize expression, more preferably referred to as plant optimizedgene(s), is to generate a DNA sequence having a G+C content preferablyclose to that of maize genes coding for metabolic enzymes. Another goalin the design of the plant optimized gene(s) encoding a bacterialprotein is to generate a DNA sequence in which the sequencemodifications do not hinder translation.

Table Ex2-1 presents the G+C content of maize genes. For the data inTable Ex2-1, coding regions of the genes were extracted from GenBank(Release 71) entries, and base compositions were calculated using theMacVector™ program (Accelerys, San Diego, Calif.). Intron sequences wereignored in the calculations.

TABLE EX 2-1 Compilation of G + C contents of protein coding regions ofmaize genes Protein Class^(a) Range % G + C Mean % G + C^(b) MetabolicEnzymes (76) 44.4-75.3 59.0 (±8.0) Structural Proteins (18) 48.6-70.563.6 (±6.7) Regulatory Proteins (5) 57.2-68.8 62.0 (±4.9)Uncharacterized Proteins (9) 41.5-70.3 64.3 (±7.2) All Proteins (108)44.4-75.3 60.8 (±.5.2)^(c) ^(a)Number of genes in class given inparentheses. ^(b)Standard deviations given in parentheses. ^(c)Combinedgroups mean ignored in mean calculation

Multiple publicly available DNA sequence databases exist wherein one mayfind information about the G+C contents of plant genomes or the proteincoding regions of various plant genes. One such database is located onthe World Wide Web at the URL http://www.kazusa.or.jp/codon/. At thissite, one may find that the average G+C content of, for example, tobacco(Nicotiana tabacum) protein coding sequences is 43.3% (analysis of 1268sequences comprising 453,797 codons). One may also find that the averageG+C content of maize (Zea mays) protein coding sequences is 54.9%(analysis of 2280 sequences comprising 973,578 codons). In comparison,the G+C content of the Sphingobium herbicidovorans AAD-13 protein codingsequence disclosed in SEQ ID NO:2 is 67.2%. Thus, it may be advantageouswhen designing an AAD-13 gene for expression in maize or dicots to lowerthe G+C content of the protein coding region to a range of 40-55%.Therefore, one goal in the design of genes encoding a bacterial proteinfor plant expression, more preferably referred to as plant optimizedgene(s), is to generate a DNA sequence having a G+C content preferablyclose to that of native host plant genes coding for metabolic enzymes.

Due to the plasticity afforded by the redundancy/degeneracy of thegenetic code (i.e., some amino acids are specified by more than onecodon), evolution of the genomes in different organisms or classes oforganisms has resulted in differential usage of redundant codons. This“codon bias” is reflected in the mean base composition of protein codingregions. For example, organisms with relatively low G+C contents utilizecodons having A or T in the third position of redundant codons, whereasthose having higher G+C contents utilize codons having G or C in thethird position. It is thought that the presence of “minor” codons withinan mRNA may reduce the absolute translation rate of that mRNA,especially when the relative abundance of the charged tRNA correspondingto the minor codon is low. An extension of this is that the diminutionof translation rate by individual minor codons would be at least anadditive for multiple minor codons. Therefore, mRNAs having highrelative contents of minor codons would have correspondingly lowtranslation rates. This rate would be reflected by subsequent low levelsof the encoded protein.

In engineering genes encoding a bacterial protein for expression inmaize (or other plants, such as cotton or soybean), it is helpful if thecodon bias of the prospective host plant(s) has been determined. Thecodon bias can be calculated as the frequency at which a single codon isused relative to the codons for all amino acids. Alternatively, asdisclosed in Table Ex2-2, Columns C, D, I and J, the codon bias may becalculated as the frequency at which a single codon is used to encode aparticular amino acid, relative to all the other codons for that aminoacid (synonymous codons). The codon bias for maize is the statisticalcodon distribution that the plant uses for coding its proteins, and thecodon usage calculated from 706 maize genes is shown in Table Ex2-2,Columns C and I. In designing coding regions for genes encodingbacterial proteins destined for plant expression, the primary (“firstchoice”) codons preferred by the plant should be determined, as well asthe second, third, fourth etc. choices of preferred codons when multiplechoices exist. A new DNA sequence can then be designed which encodes theamino sequence of the bacterial protein, but the new DNA sequencediffers from the native bacterial DNA sequence (encoding the protein) bythe substitution of plant (first preferred, second preferred, thirdpreferred, or fourth preferred, etc.) codons to specify the amino acidat each position within the protein amino acid sequence. The newsequence is then analyzed for restriction enzyme sites that might havebeen created by the modifications. The identified sites are furthermodified by replacing the codons with first, second, third, or fourthchoice preferred codons. Other sites in the sequence which could affecttranscription or translation of the gene of interest are the exon:intronjunctions (5′ or 3′), poly A addition signals, or RNA polymerasetermination signals. The sequence is further analyzed and modified toreduce the frequency of TA or CG doublets. In addition to the doublets,G or C sequence blocks that have more than about six residues that arethe same can affect transcription or translation of the sequence.Therefore, these blocks are advantageously modified by replacing thecodons of first or second choice, etc. with the next preferred codon ofchoice.

Thus, in order to design plant optimized genes encoding a bacterialprotein, a DNA sequence is designed to encode the amino acid sequence ofsaid protein utilizing a redundant genetic code established from a codonbias table compiled from the gene sequences for the particular plant orplants. The resulting DNA sequence has a higher degree of codondiversity, a desirable base composition, can contain strategicallyplaced restriction enzyme recognition sites, and lacks sequences thatmight interfere with transcription of the gene, or translation of theproduct mRNA. Such synthetic genes that are functionally equivalent tothe genes/proteins of the subject invention can be used to transformhosts, including plants. Additional guidance regarding the production ofsynthetic genes can be found in, for example, U.S. Pat. No. 5,380,831and PCT application WO 97/13402.

To engineer a plant-optimized gene encoding an AAD-13 protein, a DNAsequence was designed to encode the AAD-13 amino acid sequence,utilizing a redundant genetic code established from codon bias tablescompiled from the protein coding sequences for the particular hostplants (maize and dicots). In Table Ex2-2, Columns C, D, 1, and Jpresent the distributions (in % of usage for all codons for that aminoacid) of synonymous codons for each amino acid, as found in 706 codingregions of Zea mays (maize) and 154 dicot genes [REF: Murray, E. E.,Lotzer, J., Eberle, M. (1989) Codon usage in plant genes. Nucl. AcidsRes. 17:477-497]. The codons most preferred by each plant type areindicated in bold font, and the second, third, or fourth choices ofcodons can be identified when multiple choices exist. It is evident thatsome synonymous codons for some amino acids are found only rarely inplant genes (e.g. AGT in maize and CCG in dicots). Also, maize and dicotplants differ in individual codon usage (e.g. Alanine codon GCG occursmore frequently in maize genes than in dicot genes, while Arginine codonAGA is more often used in dicot genes than in maize genes). Thus, it isobvious that a protein coding region designed to reflect the optimalcodon composition of genes of one plant species may have a suboptimalcodon composition for expression in another plant species. In the designprocess of creating a protein-encoding DNA sequence that approximates anaverage codon distribution of both maize and dicot genes, any codon thatis used infrequently relative to the other synonymous codons for thatamino acid in either type of plant was excluded (indicated by DNU inColumns F and L of Table Ex2-2). Usually, a codon was considered to berarely used if it is represented at about 10% or less of the time toencode the relevant amino acid in genes of either plant type (indicatedby NA in Columns E and K of Table Ex2-2). To balance the distribution ofthe remaining codon choices for an amino acid, a Weighted Averagerepresentation for each codon was calculated, using the formula:

Weighted Average % of C1=1/(% C1+% C2+% C3+etc.)×% C1×100 where C1 isthe codon in question and % C2, % C3, etc. represent the % averagevalues for maize and dicots of remaining synonymous codons (% averagevalues for the relevant codons are taken from Columns E and K) of TableEx2-2.

The Weighted Average % value for each codon is given in Columns F and Lof Table Ex2-2.

TABLE Ex2-2 Synonymous codon representation in coding regions of 706 Zeamays (maize) genes (Columns C and I), and 154 dicot genes (Columns D andJ). Values for a balanced-biased codon representation set for aplant-optimized synthetic gene design are in Columns F and L. E K AMaize- F G Maize- L Amino B C D Dicot Weighted Amino H I J DicotWeighted Acid Codon Maize % Dicot % Average Average Acid Codon Maize %Dicot % Average Average ALA (A) GCA 18 25 21.7 25.5 LEU (L) CTA 8 8 NADNU GCC 34 27 30.3 35.6 CTC 26 19 22.5 34.3 GCG 24 6 NA DNU CTG 29 9 NADNU GCT 24 42 33.2 39.0 CTT 17 28 22.5 34.3 ARG (R) AGA 15 30 22.4 27.4TTA 5 10 NA DNU AGG 26 25 25.7 31.5 TTG 15 26 20.6 31.4 CGA 9 8 NA DNULYS (K) AAA 22 39 30.6 30.6 CGC 24 11 17.7 21.7 AAG 78 61 69.4 69.4 CGG15 4 NA DNU MET (M) ATG 100 100 100   100 CGT 11 21 15.8 19.4 PHE (F)TTC 71 55 63.2 63.2 ASN (N) AAC 68 55 61.4 61.4 TTT 29 45 36.8 36.8 AAT32 45 38.6 38.6 PRO (P) CCA 26 42 33.8 41.4 ASP (D) GAC 63 42 52.6 52.6CCC 24 17 20.7 25.3 GAT 37 58 47.4 47.4 CCG 28 9 NA DNU CYS (C) TGC 6856 61.8 61.8 CCT 22 32 27.2 33.3 TGT 32 44 38.2 38.2 SER (S) AGC 23 1820.4 26.0 END TAA 20 48 33.8 AGT 9 14 NA DNU TAG 21 19 20.1 TCA 16 1917.5 22.4 TGA 59 33 46.1 TCC 23 18 20.6 26.3 GLN (Q) CAA 38 59 48.4 48.4TCG 14 6 NA DNU CAG 62 41 51.6 51.6 TCT 15 25 19.9 25.4 GLU (E) GAA 2949 38.8 38.8 THR (T) ACA 21 27 23.8 28.0 GAG 71 51 61.2 61.2 ACC 37 3033.6 39.5 GLY (G) GGA 19 38 28.5 28.5 ACG 22 8 NA DNU GGC 42 16 29.129.0 ACT 20 35 27.7 32.5 GGG 20 12 16.1 16.0 TRP (W) TGG 100 100 100  100   GGT 20 33 26.7 26.6 TYR (Y) TAC 73 57 65.0 65.0 HIS (H) CAC 62 4654.1 54.1 TAT 27 43 35.0 35.0 CAT 38 54 45.9 45.9 VAL (V) GTA 8 12 NADNU ILE (I) ATA 14 18 15.9 15.9 GTC 32 20 25.8 28.7 ATC 58 37 47.6 47.9GTG 39 29 34.1 38.0 ATT 28 45 36.4 36.4 GTT 21 39 29.9 33.3

A new DNA sequence which encodes essentially the amino acid sequence ofthe Sphingobium herbicidovorans AAD-13 protein of SEQ ID NO:2 wasdesigned for optimal expression in both maize and dicot cells using abalanced codon distribution of frequently used codons found in maize anddicot genes.

2.2—AAD-13 Plant Rebuild Analysis.

Extensive analysis of the 861 base pairs (bp) of the coding region ofthe native DNA sequence of AAD-13 (SEQ ID NO:1) revealed the presence ofseveral sequence motifs that are thought to be detrimental to optimalplant expression, as well as a non-optimal codon composition. Theprotein encoded by SEQ ID NO:1 (AAD-13) is presented as SEQ ID NO:2. Toimprove production of the recombinant protein in maize as well asdicots, a “plant-optimized” DNA sequence (AAD-13 v1) (SEQ ID NO:3) wasdeveloped that encodes a protein (SEQ ID NO:4) which is the same as thenative protein disclosed in SEQ ID NO:2 except for the addition of analanine residue at the second position (underlined in SEQ ID NO:4). Theadditional alanine codon (GCT; underlined in SEQ ID NO:3) encodes partof an Neo I restriction enzyme recognition site (CCATGG) spanning theATG translational start codon. Thus, it serves the dual purpose offacilitating subsequent cloning operations while improving the sequencecontext surrounding the ATG start codon to optimize translationinitiation. The proteins encoded by the native and plant-optimized (v1)coding regions are 99.3% identical, differing only at amino acid number2. In contrast, the native and plant-optimized (v1) DNA sequences of thecoding regions are only 77.3% identical. Table Ex2-3 shows thedifferences in codon compositions of the native (Columns A and D) andplant-optimized sequences (Columns B and E), and allows comparison to atheoretical plant-optimized sequence (Columns C and F) that would haveprecisely the codon composition dictated by columns F and L of TableEx2-2.

TABLE Ex2-3 Codon composition comparisons of coding regions of NativeAAD-13, Plant-Optimized version (v1) and a Theoretical Plant-Optimizedversion. B C E F Amino A Plant Opt Theor. Plant Amino D Plant Opt Theor.Plant Acid Codon Native v1 # Opt. # Acid Codon Native v1 # Opt. # ALA(A) GCA 1 10 9 LEU (L) CTA 0 0 0 GCC 24 11 13 CTC 11 11 10 GCG 10 0 0CTG 17 0 0 GCT 1 16 14 CTT 0 10 10 ARG (R) AGA 0 4 4 TTA 0 0 0 AGG 0 5 5TTG 2 9 9 CGA 1 0 0 LYS (K) AAA 0 3 3 CGC 10 4 3 AAG 10 7 7 CGG 4 0 0MET (M) ATG 9 9 9 CGT 1 3 3 PHE (F) TTC 8 6 6 ASN (N) AAC 3 2 2 TTT 1 33 AAT 1 2 2 PRO (P) CCA 2 7 7 ASP (D) GAC 19 13 13 CCC 5 5 5 GAT 5 11 11CCG 10 0 0 CYS (C) TGC 2 1 1 CCT 1 6 6 TGT 0 1 1 SER (S) AGC 9 4 4 ENDTAA 0 0 AGT 1 0 0 TAG 0 0 TCA 1 3 3 TGA 1 1 1 TCC 1 4 4 GLN (Q) CAA 0 77 TCG 3 0 0 CAG 14 7 7 TCT 0 4 4 GLU (E) GAA 3 5 5 THR (T) ACA 0 3 3 GAG11 9 9 ACC 7 4 4 GLY (G) GGA 1 6 6 ACG 4 0 0 GGC 16 6 6 ACT 0 4 4 GGG 33 3 TRP (W) TGG 7 7 7 GGT 1 6 6 TYR (Y) TAC 5 4 5 HIS (H) CAC 7 7 8 TAT2 3 2 CAT 7 7 6 VAL (V) GTA 0 0 0 ILE (I) ATA 0 2 2 GTC 6 4 4 ATC 10 5 5GTG 7 6 6 ATT 1 4 4 GTT 2 5 5 Totals 157 158 158 Totals 131 131 131

It is clear from examination of Table Ex2-3 that the native andplant-optimized coding regions, while encoding nearly identicalproteins, are substantially different from one another. ThePlant-Optimized version (v1) closely mimics the codon composition of atheoretical plant-optimized coding region encoding the AAD-13 protein.

2.3 Rebuild for E. coli Expression

Specially engineered strains of Escherichia coli and associated vectorsystems are often used to produce relatively large amounts of proteinsfor biochemical and analytical studies. It is sometimes found that anative gene encoding the desired protein is not well suited for highlevel expression in E. coli, even though the source organism for thegene may be another bacterial organism. In such cases it is possible anddesirable to re-engineer the protein coding region of the gene to renderit more suitable for expression in E. coli. E. coli Class II genes aredefined as those that are highly and continuously expressed during theexponential growth phase of E. coli cells. [REF: Henaut. A. and Danchin,A. (1996) in Escherichia coli and Salmonella typhimurium cellular andmolecular biology, vol. 2, pp. 2047-2066. Neidhardt, F., Curtiss III. R.Ingraham, J. Lin, E., Low, B., Magasanik, B. Reznikoff, W., Riley, M.,Schaechter, M. and Umbarger, H. (eds.) American Society forMicrobiology. Washington, D.C.]. Through examination of the codoncompositions of the coding regions of E. coli Class II genes, one candevise an average codon composition for these E. coli Class II genecoding regions. It is thought that a protein coding region having anaverage codon composition mimicking that of the Class II genes will befavored for expression during the exponential growth phase of E. coli.Using these guidelines, a new DNA sequence that encodes the AAD-13protein (SEQ ID NO:4; including the additional alanine at the secondposition, as mentioned above), was designed according to the averagecodon composition of E. coli Class II gene coding regions. The initialsequence, whose design was based only on codon composition, was furtherengineered to include certain restriction enzyme recognition sequencessuitable for cloning into E. coli expression vectors. Detrimentalsequence features such as highly stable stemloop structures wereavoided, as were intragenic sequences homologous to the 3′ end of the16S ribosomal RNA (i.e. Shine Dalgarno sequences) The E. coli-optimizedsequence (v2) is disclosed as SEQ ID NO:5 and encodes the proteindisclosed in SEQ ID NO:4.

The native and E. coli-optimized (v2) DNA sequences are 80.2% identical,while the plant-optimized (v1) and E. coli-optimized (v2) DNA sequencesare 84.4% identical. Table Ex2-4 presents the codon compositions of thenative AAD-13 coding region; Columns A and D), the AAD-13 coding regionoptimized for expression in E. coli (v2; Columns B and E) and the codoncomposition of a theoretical coding region for the AAD-13 protein havingan optimal codon composition of E. coli Class II genes (Columns C andF).

TABLE Ex2-4 Codon composition comparisons of coding regions of NativeAAD-13, E. coli-Optimized version (v2) and a Theoretical E. coli ClassII-Optimized version. B C E F Amino A E. coli Theor. Amino D E. coliTheor, Acid Codon Native Opt v2 # Class II # Acid Codon Native Opt v2 #Class II # ALA (A) GCA 1 11 11 LEU (L) CTA 0 0 0 GCC 24 0 0 CTC 11 0 0GCG 10 14 14 CTG 17 30 30 GCT 1 12 12 CTT 0 0 0 ARG (R) AGA 0 0 0 TTA 00 0 AGG 0 0 0 TTG 2 0 0 CGA 1 0 0 LYS (K) AAA 0 8 8 CGC 10 7 5 AAG 10 22 CGG 4 0 0 MET (M) ATG 9 9 9 CGT 1 9 11 PHE (F) TTC 8 6 6 ASN (N) AAC 34 4 TTT 1 3 3 AAT 1 0 0 PRO (P) CCA 2 3 3 ASP (D) GAC 19 13 13 CCC 5 0 0GAT 5 11 11 CCG 10 15 15 CYS (C) TGC 2 1 1 CCT 1 0 0 TGT 0 1 1 SER (S)AGC 9 4 4 END TAA 0 1 1 AGT 1 0 0 TAG 0 0 0 TCA 1 0 0 TGA 1 0 0 TCC 1 55 GLN (Q) CAA 0 3 3 TCG 3 0 0 CAG 14 11 11 TCT 0 6 6 GLU (E) GAA 3 10 11THR (T) ACA 0 0 0 GAG 11 4 3 ACC 7 7 7 GLY (G) GGA 1 0 0 ACG 4 0 0 GGC16 10 10 ACT 0 4 4 GGG 3 0 0 TRP (W) TGG 7 7 7 GGT 1 11 11 TYR (Y) TAC 55 5 HIS (H) CAC 7 10 10 TAT 2 2 2 CAT 7 4 4 VAL (V) GTA 0 3 3 ILE (I)ATA 0 0 0 GTC 6 0 0 ATC 10 7 7 GTG 7 5 5 ATT 1 4 4 GTT 2 7 7 Totals 157158 158 Totals 131 131 131

It is clear from examination of Table Ex2-4 that the native and E.coli-optimized coding regions, while encoding nearly identical proteins,are substantially different from one another. The E. coli-Optimizedversion (v2) closely mimics the codon composition of a theoretical E.coli-optimized coding region encoding the AAD-13 protein.

Example 3 Cloning of Expression and Transformation Vectors

3.1 Construction of E. coli, pET Expression Vector.

Using the restriction enzymes corresponding to the sites added with theadditional cloning linkers (Xba 1, Xho 1) AAD-13 (v2) was cut out of thepicoscript vector, and ligated into a pET280 streptomycin/spectinomycinresistant vector. Ligated products were then transformed into TOP10F′ E.coli, and plated on to Luria Broth+50 μg/ml Streptomycin & Spectinomycin(LB S/S) agar plates.

To differentiate between AAD-13 (v2):pET280 and pCR2.1:pET280 ligations,approximately 20 isolated colonies were picked into 6 ml of LB-S/S, andgrown at 37° C. for 4 hours with agitation. Each culture was thenspotted onto LB+Kanamycin 50 μg/ml plates, which were incubated at 37°C. overnight. Colonies that grew on the LB-K were assumed to have thepCR2.1 vector ligated in, and were discarded. Plasmids were isolatedfrom the remaining cultures as before, and checked for correctness withdigestion by Fsp1. The final expression construct was given thedesignation pDAB4115.

3.3—Completion of Binary Vectors.

The plant optimized gene AAD-13 (v1) was received from Picoscript (thegene rebuild design was completed (see above) and out-sourced toPicoscript for construction) The AAD-13 (v1) gene was cloned intopDAB4055 as an Nco I-Sac I fragment. The resulting construct wasdesignated pDAB4113, containing: [AtUbi10 promoter: AAD-13 (v1): AtuORF13′UTR] (verified with Nco I and Sac I restriction digests). A Not I-NotI fragment containing the described cassette was then cloned into theNot I site of the binary vector pDAB3038. The resulting binary vector,pDAB4114, containing the following cassette [AtUbi10 promoter: AAD-13(v1): AtuORF1 3′UTR: CsVMV promoter: PAT: ORF25/26 3′UTR] wasrestriction digested (with SacI) for verification of the correctorientation. The verified completed construct (pDAB4114) was used fortransformation into Agrobacterium (see Example 6).

Example 4 Recombinant AAD-13 Expression and Purification in Pseudomonasfluorescens

4.1—Pseudomonas fluorescens Fermentation

For shake flask experiment, 200 μl of the Pseudomonas fluorescens strainglycerol stock carrying the AAD-13 (v1) construct (sec 3.2) will be usedto inoculate 50 ml fresh LB media supplemented with 30 μg/mltetracycline/HCl. The culture (in a 250 ml baffled Erlenmeyer flask)will be incubated on a shaker (New Brunswick Scientific Model Innova 44)at 300 rpm and 30° C. for 16 hrs. 20 ml of seed culture will betransferred into 1 L Pseudomonas fluorescens culture media (Yeastextract, 5 g/L; K₂HPO₄, 5 g/L; (NH₄)₂PO₄, 7.5 g/L; (NH₄)₂SO₄;MgSO₄.7H₂O, 1 g/L; KCl, 0.5 g/L; CaCl₂-2H₂O, 0.5 g/L; NaCitrate-2H₂O, 15g/L; Glycerol, 95 g/L; Trace element solution, 10 ml/L; Trace elementsolution: FeCl₃-6H₂O, 5.4 g/L; MnCl₂-4H₂O, 1 g/L; ZnSO₄.7H₂O, 1.45 g/L;CuSO₄.5H₂O, 0.25 g/L; H₃BO₃, 0.1 g/L; (NH₄)₆MO₇O₂₄, 0.1 g/L;concentrated HCl, 13 ml/L) supplemented with 20 μg/ml tetracycline/HCland 250 μl of Pluronic L61 (anti-foam) in a 2.8 L baffled Erlenmeyerflask. The cultures are to be incubated at 30° C. and 300 rpm for 24hrs. Isopropyl β-D-1-thiogalacto-pyranoside (IPTG) will be added to 1 mMfinal in the cultures and continued to incubate for approximately 48 hrsat 25° C. Cells are harvested by centrifugation at 7 krpm at 4° C. for15 min, and cell paste is stored at −80° C. or immediately processed forpurification.

For tank experiments, 1 ml each of the glycerol stock will be inoculateda 1 L baffled flask containing 200 ml of LB media supplemented with 30μg/ml tetracycline/HCl at 300 rpm and 32° C. for 16-24 hrs. The combinedculture from three flasks (600 ml) is then aseptically transferred to a20 L fermentor (B. Braun Bioreactor Systems) containing 10 L of Dowproprietary defined medium (through Teknova, Hollister, Calif.) designedto support high cell density growth. Growth temperature is maintained at32° C. and the pH is controlled at the desired set-point through theaddition of aqueous ammonia. Dissolved oxygen will be maintained at apositive level in the liquid culture by regulating the sparged air flowand the agitation rates. The fed-batch fermentation process is carriedout for approximately 24 hrs till cell density reaches 170-200 OD₅₇₅.IPTG is then added to 1 mM to induce the recombinant protein expressionand the temperature is reduced and maintained at 25° C. usingcirculation of cold-water supply. The induction phase of thefermentation will be allowed to continue for another 24 hrs. Samples (30ml) are collected for various analyses to determine cell density andprotein expression level at 6, 12, and 18 hrs post-induction timepoints. At the end of a fermentation run, cells are harvested bycentrifugation at 10 krpm for 30 min. The cell pellets are then frozenat −80° C. for further processing.

4.2—Purification of AAD-13 for Biochemical Characterization and AntibodyProduction

Approximately 100-200 g of frozen (or fresh) Pseudomonas cells arethawed and resuspended in 1-2 L of extraction buffer containing 20 mMTris-HCl, pH 8.5, and 25 ml of Protease inhibitor cocktail (Sigmacat#P8465). The cells are disrupted using Microfluidizer (model M110L or110Y) (Microfluidics, Newton, Mass.) on ice with one pass at11,000-12,000 psi. The lysate is centrifuged at 24,000 rpm for 20 min.The supernatant will be transferred and dialyzed against 10 volumes of20 mM Tris-HCl, pH 8.5 overnight at 4° C., or diafiltrated with thisbuffer and filtered through a 0.45 μm membrane before applying to thecolumn separations. All subsequent protein separations will be performedusing Pharmacia AKTA Explorer 100 and operated at 4° C. Prior toloading, a Q Sepharose Fast Flow column (Pharmacia XK 50/00, 500 ml bedsize) is equilibrated with 20 mM Tris-HCl, pH 8.5 buffer. The sample isapplied to the column at 15 ml/min and then washed with this bufferuntil the eluate OD₂₈₀ returned to baseline. Proteins are eluted with 2L of linear gradient from 0 to 0.3 M NaCl at a flow rate of 15 ml/min,while 45 ml fractions are collected. Fractions containing AAD-13activity as determined by the colorimetric enzyme assay and alsocorresponding to the predicted molecular weight of AAD-13 protein (about32 kDa band on SDS-PAGE) are to be pooled. Solid ammonium sulfate tofinal 0.5 M is added to the sample, and then applied to a Phenyl HPcolumn (Pharmacia XK 50/20, 250 ml bed size) equilibrated in 0.5 Mammonium sulfate in 20 mM Tris-HCl, pH 8.0. This column is washed withthe binding buffer at 10 ml/min until the OD₂₈₀ of the eluate returnedto baseline, proteins are eluted within 2 column volumes at 10 ml/min bya linear gradient from 0.5 M to 0 Ammonium sulfate in 20 mM Tris-HCl, pH8.0, and 12.5 ml fractions are collected. The main peak fractionscontaining AAD-13 will be pooled, and if necessary, concentrated using aMWCO 10 kDa cut-off membrane centrifugal filter device (Millipore). Insome cases the sample is further applied to a Superdex 75 gel filtrationcolumn (Pharmacia XK 16/60, 110 ml bed size) with PBS buffer at a flowrate of 1 ml/min. Peak fractions containing pure AAD-13 are pooled andstored at −80° C. for future use.

Example 5 In Vitro Assays of AAD-13 Activity

5.1—Assay Via Colorimetric Phenol Detection.

Enzyme activity will be measured by colorimetric detection of theproduct phenol using a protocol modified from that of Fukumori andHausinger (1993) (J. Biol. Chem. 268: 24311-24317) to enable deploymentin a 96-well microplate format. The colorimetric assay has beendescribed for use in measuring the activity of dioxygenases cleaving2,4-D and dichlorprop to release the product 2,4-dichlorophenol. Thecolor yield from several phenols was compared to that of2,4-dichlorophenol using the detection method previously described toascertain which phenol products could be readily detected. Phenols andphenol analogs were tested at a final concentration of 100 μM in 0.15 ml20 mM MOPS pH 6.75 containing 200 μM NH₄(FeSO₄)₂, 200 μM sodiumascorbate. Pyridinols derived from fluoroxypyr and triclopyr produced nosignificant color. The color yield of 2,4-dichlorophenol was linear andproportional to the concentration of phenol in the assay up to ˜500 μM.A calibration curve performed under standard assay conditions (160 μlfinal assay volume) indicated that an absorbance at 510 nm of 0.1 wasobtained from 17.2 μM phenol.

Enzyme assays are performed in a total volume of 0.16 ml 20 mM MOPS pH6.75 containing 200 μM NH₄FeSO₄, 200 μM sodium ascorbate, 1 mMα-ketoglutarate, the appropriate substrate (added from a 100 mM stockmade up in DMSO), and enzyme. Assays are initiated by addition of thearyloxyalkanoate substrate, enzyme or α-ketoglutarate at time zero.After 5 minutes of incubation at 25° C., the reaction is terminated byaddition of 30 μl of a 1:1:1 mix of 50 mM Na EDTA; pH 10 buffer (3.09 gboric acid+3.73 g KCl+44 ml 1 N KOH) and 0.2% 4-aminoantipyrine. Then 10μl 0.8% potassium ferricyanide is added and after 5 or 10 min, theabsorbance at 510 nm was recorded in a spectrophotometric microplatereader. Blanks contained all reagents except for enzyme to account forthe occasional slight contamination of some of the substrates by smallamounts of phenols.

5.2—Assay Via Detection of Chloropyridinol

AAD-13 action on potential substrates such as the herbicide triclopyrcontaining a substituted pyridine (rather than benzene rings) willrelease a pyridinol on cleavage of the aryloxyalkanoate bond. Pyridinolswere not detected using the aminoantipyrine/ferricyanide phenoldetection described in the preceding section. However, it was found thatproduct chloropyridinols absorb strongly in the near UV with λ_(max) of325 nm at pH 7 (extinction coefficient ˜8,400 M⁻¹·cm⁻¹). This was usedto create a continuous microplate-based spectrophotometric assay. Assaysare performed in a total volume of 0.2 ml 20 mM MOPS pH 6.75 containing200 μM NH₄FeSO₄, 200 μM sodium ascorbate, 1 mM α-ketoglutarate, theappropriate substrate (added from a 100 mM stock made up in DMSO), andenzyme. Assays are initiated by addition of the aryloxyalkanoatesubstrate, enzyme or α-ketoglutarate at time zero and the increase inabsorbance followed for 10 minutes at 325 nm in a microplate reader. Thefirst 2 minutes of the reaction will be used to determine initial rates.

5.3—Calorimetric Assay Using 2-(2-chloro,4-nitrophenoxy)propionate

A convenient assay of AAD-13 was devised using2-(2-chloro,4-nitrophenoxy)propionate (CNPP) as substrate. Cleavage ofCNPP by AAD-13 will release 2-chloro,4-nitrophenol. This phenol has abright yellow absorbance at 410 nm at pH 7 enabling the reaction to befollowed continuously or by endpoint analysis. The presence of AAD-13activity can be monitored visually without the need for addition offurther reagents. Microplate-based spectrophotometric assays wereperformed in a total volume of 0.2 ml 20 mM MOPS pH 6.75 containing 200μM NH₄FeSO₄, 200 μM sodium ascorbate, 1 mM α-ketoglutarate, theappropriate amount of CNPP (added from a 10 mM stock made up in DMSO),and enzyme. Assays are initiated by addition of CNPP, enzyme, orα-ketoglutarate at time zero and the increase in absorbance followed for10 min at 410 nm in a microplate reader. The first 2 min of the reactionwill be used to determine initial rates. A calibration curve performedunder standard assay conditions (200 μl final assay volume) indicatedthat an absorbance at 410 nm of 0.1 was obtained from 25.1 μM 2-chloro,4-nitrophenol. Using this assay, the kinetic constants for CNPP as asubstrate were determined to be K_(m)=31±5.5 μM and k_(cat)=16.2±0.79min⁻¹.

5.4—Coupled Assay

In order to test a broad range of substrates, the production ofsuccinate from the breakdown of α-ketoglutarate was detectedspectrophotometricly using a protocol based on the method of Luo et. al.(2006) (Anal. Biochem. 353: 69-74). As depicted in FIG. 3, theconcomitant breakdown of α-ketoglutarate and the substrate of interestvia AAD-13, results in the production of succinate. Succinate is furthermodified to succinyl-CoA by succinyl-CoA synthetase which consumes ATPand produces ADP. ADP is then consumed by the commonly employed pyruvatekinase/lactate dehydrogenase enzymatic coupling system (Sigma P0294).The resulting conversion of NADH to NAD is monitoredspectrophotometrically at 340 nm.

5.4.1—Cloning and Expression of His-Tagged Succinyl-CoA Synthetase andAAD-13 (v2)

The two E. coli genes that encode the synthetase, sucC and sucD, wereamplified out of the Top10 strain of E. coli from Invitrogen as a singleamplicon. Genomic DNA was obtained by boiling an aliquot of cells for 10min, then centrifuging, and retaining the supernatant containing theDNA. As template for AAD-13 (v2), the previously created pET clonepDAB4115 was used. To amplify the sucCD genes, the following primerswere used: suc-Nde (SEQ ID 9) 5′ CATATGAACTTACATGAATATCAGGCAAAAC 3′ andsuc-Xho (SEQ ID 10) 5′ CTCGAGTTTCAGAACAGTTTTCAGTGCTTC 3′. For AAD-13(v2), the following primers were used: aad-13F (SEQ ID 11) 5′CATATGGCGAGCCCGGCG 3′ and aad-13R (SEQ ID 12) 5′CTCGAGGTGTGCCAGTGCGGTCTC 3′. These add suitable restriction sites fordownstream cloning and remove the stop codon to permit His-tagging. Forthe reaction, thermal cycler conditions were: 96° C. 2 min, then 35cycles of: 96° C. 30 sec, 53° C. 30 sec, 72° C. 1.5 min, followed by aone final cycle of 72° C. 5 min. The resulting amplicons were sub-clonedto verify correct sequence. Clones for each containing the correctinsert were digested with Nde1/Xho1 and the inserts were then clonedinto the pET-26b(+) expression vector. For expression, a lawn oftransformed BL-21 E. coli was scraped into 50 ml of LB+Kan (50 μg/ml)and grown at 37° C. for 2 hrs. Two millilers of this culture weretransferred into 100 ml of LB+Kan. These flaskes were grown at 37° C.for 4 hrs. Cells were induced with 50 μM IPTG, and grown overnight at25° C. Cultures were centrifuged, and cell pellet used for proteinpurification.

5.4.2—Purification of AAD-13 and his-Tagged Succinyl CoA Synthetase forIn Vitro Substrate Identification

His-tagged AAD-13 was purified using metal affinity chromatographyprotocols based on the column manufacturer's directions. Cell pelletsharvested from 1 L of culture and stored at −80° C. were thawed andresuspended in 20 mL of extraction buffer (100 mM Tris-HCl, pH 8;200-300 μL protease inhibitor cocktail (Sigma P8849), 1 mg/mL lysozyme,and 1 mM MgCl₂). Resuspended cells incubated at room temperature for10-15 min prior to treating with DNase to reduce viscosity. Allsubsequent steps were carried out at 4° C. The extract was centrifugedfor 20 min at 20,000×g to clarify. Using a flow rate of 1 mL/min, theresulting supernatant was applied to 2 consecutive 1 mL Co-MAC™Cartridges (EMD/Novagen 71650) previously equilibrated with buffer A (25mM Tris pH 8.0, 0.5 M NaCl). After the extract was loaded, the columnwas washed with 5 mM imidazole in buffer A until the OD₂₈₀ returned tobaseline. Protein was eluted with 50 mM imidazole in buffer A. Fractionscontaining predominantly AAD-13 as indicated by an approximately 30 kDaband on SDS-PAGE were exchanged into buffer C (20 mM Tris pH 8.0, 100 mMNaCl, 2 mM DTT) using BG-10 desalting columns (Bio-Rad). AAD-13 inbuffer C was then assayed spectrophotometrically according to the invitro coupled assay.

His-tagged succinyl CoA synthetase was purified utilizing consecutive 1mL Co-MAC™ Cartridges (EMD/Novagen 71650) and protocols based on themanufacturer's directions. Cell pellets that had been stored at −80° C.were thawed and resuspended in 50 mL of extraction buffer (100 mM TrispH 7.2, 200-300 μL protease inhibitor cocktail (Sigma P8849), 1 mg/mLlysozyme, and 1 mM MgCl₂) per L of cell culture. Resuspended cells wereincubated at room temperature for 10-15 min prior to treating with DNaseto reduce viscosity. All subsequent steps were carried out at 4° C.unless noted otherwise. The extract was centrifuged for 20 min at20,000×g to clarify. At this point, supernatant can either be applieddirectly to Co-MAC™ Cartridges pre-equilibrated with binding buffer(0.5M NaCl, 20 mM Tris-HCl pH 7.9 and 5 mM imidazole) or brought to 80%ammonium sulfate. The ammonium sulfate treated sample was centrifugedfor 20 min at 20,000×g to pellet protein. Pellet was resuspended inbuffer A (20 mM Tris-HCl pH 8.0 and 0.5M NaCl) and residual ammoniumsulfate was removed using BG-10 desalting columns (Bio-Rad)pre-equilibrated with buffer A. The resulting samples were applied toCo-MAC™ Cartridges pre-equilibrated with binding buffer and a flow rateof 1 mL/min. Following application of extracted protein, column wasrinsed with 10 column volumes of 0.5% buffer B (20 mM Tris-HCl, 0.5MNaCl, and 1 M imidazole). This was followed by a 5 column volume stepgradient of 6% buffer B and an additional 10 column volume step gradientof 50% buffer B. The majority of the desired protein eluted with the 6%buffer B gradient. Fractions containing succinyl CoA synthetase wereidentified by the presence of two bands corresponding to the succinylCoA synthetase subunits (˜40 & 33 kDa) via SDS PAGE and the detection ofcorresponding in vitro activity. Succinyl CoA synthetase activity wasconfirmed using a modified version of the in vitro coupled assay below.Briefly, reaction progress was monitored spectrophotometrically at 340nm in the presence of 100 mM tris pH 8.0, 1 mM PEP 0.4 mM NADH 10 mMMgCl₂, 0.2 mM CoA, 0.2 mM ATP, 3.5 U/mL PK, 5 U/mL LDH, and SCS.Reaction was initiated by the addition of 1 mM succinate.

5.4.3—In Vitro Coupled Assay

Identification of AAD-13 (v2) substrates in vitro was based on enzymaticactivity detected during continuous spectrophotometric monitoring of a0.2 mL reaction volume in a 96 well microtiter plate. Reactionconditions were as follows: 100 mM MOPS pH 7.0, 0.4 mM NADH, 0.4 mM ATP,0.4 mM CoA, 1 mM PEP, 10 mM MgCl₂, 0.1 mM FeSO₄ (solubilized in HCl),and 0.1 mM ascorbate, 1 mM α-ketoglutarate and sufficient AAD-13 (v2) toproduce an observable rate in the presence of 2,4-D. Coupling enzymes(SCS/PK/LDH) were adjusted by batch to ensure adequate coupling, andpotential substrates were generally assayed at 1 mM. Alterations insubstrate concentrations were made as needed to adjust for solubility.Reactions were initiated by either the addition of AAD-13 (v2) orpotential substrate. The rate of substrate independent conversion ofα-ketoglutarate to succinate by AAD was monitored under the above assayconditions and subtracted from the observed reaction rates. Reactionrates observed with propionate substrates were divided by two to adjustfor the production of pyruvate resulting from the cleavage of thesecompounds via AAD. Additionally, propionate compounds were checked forpyruvate contamination by spectrophotometrically monitoring theconsumption of NADH in the presence of compound and PK/LDH.

5.4.4—In Vitro Screening Results

Table Ex5 displays the AAD-13 (v2) reaction rate observed with multiplechemistries via the in vitro coupled assay. Reaction rates are reportedas a percentage of the 2,4-D reaction rate obtained in the same sampleset. This data can be used to qualitatively segregate substrates fromnon-substrates, as well as identify trends in substrate efficiency. Itshould be noted that faster rates can be more difficult to accuratelycompare depending on the percentage of available substrate consumed.This is particularly true of propionate compounds which display twicethe rate as non-propionate compounds for the equivalent number of enzymeturnovers. As a result, highly efficient substrates will be properlygrouped when compared to low efficiency substrates. Within the groupingof highly efficient substrates however, compounds may not bequantitatively separated by a screen using single rates of substrate andAAD. Compounds denoted with an asterisk were tested at 0.5 mM instead of1 mM due to absorbance interference at higher concentrations.

TABLE Ex5 % of substrate 24D MOL. Name X # Y/N activity STRUCTURE 191716y 66

571320 y 39

93116 y 128

475726 y 112

118942 y 46

470901 y 30

R-fenoxaprop 11044492 N 2

Mecoprop 188874 y 169

r dichlorprop 19 r,s dichlorprop 117613 Y 195

S-dichlorprop y 233 2,4-D 195517 y 100

24DB 178577 N 2

3-amino 24D 11263526 y 151

11113675 y 113

124988 y 44

83293 y 106

alpha methyl floroxypyr 11182286 y 43

fluoroxypyr 68316 y 67

triclopyr 156136 N 6

93833 y 33

66357 y 24

91767 y 88

116844 y 25

diclofop 460511 y >100

fluazifiop 67131 y ~50

quizalofop 44936* Y

cyhalofop 7466 y

66732 y >100

8563 y 64

193908 y 56

761310* Not Detected

11077344* Not Detected

198167 Not Detected

11077347* Not Detected

238166* Not Detected

657338 N 5

657339 N 5

11213586 N 2

11453845 N 13

187507 N 10

204558* Not Detected

188495 M 19

187439 Not Detected

1190305 Not Detected

AAD-13 is unlike other reported a-ketoglutarate-dependent dioxygenaseswho have 2,4-D-degrading activity. A key distinction is the broad arrayof aryloxy and alkyloxy-alkanoate substrates, buta number ofpyridyloxysubstitutes are effective herbicides and substrates (e.g.,fluoroxypyr) but other herbicides like triclopyr are considerably poorersubstrates. This creates a new opportunity to use alternative herbicidesfor control of transgenic plants with AAD-13 substrates. It alsoprovides opportunity to complement similar genes in planta to broadentolerance or improve the breadth of substrates to which the plants aretolerant.

Example 6 Transformation into Arabidopsis and Selection

6.1—Arabidopsis thaliana Growth Conditions.

Wildtype Arabidopsis seed was suspended in a 0.1% Agarose (SigmaChemical Co., St. Louis, Mo.) solution. The suspended seed was stored at4° C. for 2 days to complete dormancy requirements and ensuresynchronous seed germination (stratification).

Sunshine Mix LP5 (Sun Gro Horticulture, Bellevue, Wash.) was coveredwith fine vermiculite and sub-irrigated with Hoagland's solution untilwet. The soil mix was allowed to drain for 24 hours. Stratified seed wassown onto the vermiculite and covered with humidity domes (KORDProducts, Bramalea, Ontario, Canada) for 7 days.

Seeds were germinated and plants were grown in a Conviron (modelsCMP4030 and CMP3244, Controlled Environments Limited, Winnipeg,Manitoba, Canada) under long day conditions (16 hours light/8 hoursdark) at a light intensity of 120-150 mmol/m²sec under constanttemperature (22° C.) and humidity (40-50%). Plants were initiallywatered with Hoagland's solution and subsequently with deionized waterto keep the soil moist but not wet.

6.2—Agrobacterium Transformation.

An LB+agar plate with erythromycin (Sigma Chemical Co., St. Louis, Mo.)(200 mg/L) or spectinomycin (100 mg/L) containing a streaked DH5α colonywas used to provide a colony to inoculate 4 ml mini prep cultures(liquid LB+erythromycin). The cultures were incubated overnight at 37°C. with constant agitation. Qiagen (Valencia, Calif.) Spin Mini Preps,performed per manufacturer's instructions, were used to purify theplasmid DNA.

Electro-competent Agrobacterium tumefaciens (strains Z707s, EHA101s, andLBA4404s) cells were prepared using a protocol from Weigel andGlazebrook (2002). The competent Agrobacterium cells were transformedusing an electroporation method adapted from Weigel and Glazebrook(2002). 50 μl of competent agro cells were thawed on ice, and 10-25 ngof the desired plasmid was added to the cells. The DNA and cell mix wasadded to pre-chilled electroporation cuvettes (2 mm). An EppendorfElectroporator 2510 was used for the transformation with the followingconditions, Voltage: 2.4 kV, Pulse length: 5 msec.

After electroporation, 1 ml of YEP broth (per liter: 10 g yeast extract,10 g Bacto-peptone, 5 g NaCl) was added to the cuvette, and the cell-YEPsuspension was transferred to a 15 ml culture tube. The cells wereincubated at 28° C. in a water bath with constant agitation for 4 hours.After incubation, the culture was plated on YEP+agar with erythromycin(200 mg/L) or spectinomycin (100 mg/L) and streptomycin (Sigma ChemicalCo., St. Louis, Mo.) (250 mg/L). The plates were incubated for 2-4 daysat 28° C.

Colonies were selected and streaked onto fresh YEP+agar witherythromycin (200 mg/L) or spectinomycin (100 mg/L) and streptomycin(250 mg/L) plates and incubated at 28° C. for 1-3 days. Colonies wereselected for PCR analysis to verify the presence of the gene insert byusing vector specific primers. Qiagen Spin Mini Preps, performed permanufacturer's instructions, were used to purify the plasmid DNA fromselected Agrobacterium colonies with the following exception: 4 mlaliquots of a 15 ml overnight mini prep culture (liquid YEP+erythromycin(200 mg/L) or spectinomycin (100 mg/L)) and streptomycin (250 mg/L))were used for the DNA purification. An alternative to using Qiagen SpinMini Prep DNA was lysing the transformed Agrobacterium cells, suspendedin 10 μl of water, at 100° C. for 5 minutes. Plasmid DNA from the binaryvector used in the Agrobacterium transformation was included as acontrol. The PCR reaction was completed using Taq DNA polymerase fromTakara Minis Bio Inc. (Madison, Wis.) per manufacturer's instructions at0.5× concentrations. PCR reactions were carried out in a MJ ResearchPeltier Thermal Cycler programmed with the following conditions; 1) 94°C. for 3 minutes, 2) 94° C. for 45 seconds, 3) 55° C. for 30 seconds, 4)72° C. for 1 minute, for 29 cycles then 1 cycle of 72° C. for 10minutes. The reaction was maintained at 4° C. after cycling. Theamplification was analyzed by 1% agarose gel electrophoresis andvisualized by ethidium bromide staining. A colony was selected whose PCRproduct was identical to the plasmid control.

6.3—Arabidopsis Transformation.

Arabidopsis was transformed using the floral dip method. The selectedcolony was used to inoculate one or more 15-30 ml pre-cultures of YEPbroth containing erythromycin (200 mg/L) or spectinomycin (100 mg/L) andstreptomycin (250 mg/L). The culture(s) was incubated overnight at 28°C. with constant agitation at 220 rpm. Each pre-culture was used toinoculate two 500 ml cultures of YEP broth containing erythromycin (200mg/L) or spectinomycin (100 mg/L) and streptomycin (250 mg/L) and thecultures were incubated overnight at 28° C. with constant agitation. Thecells were then pelleted at approx. 8700×g for 10 minutes at roomtemperature, and the resulting supernatant discarded. The cell pelletwas gently resuspended in 500 ml infiltration media containing: ½×Murashige and Skoog salts/Gamborg's B5 vitamins, 10% (w/v) sucrose,0.044 μM benzylamino purine (10 μl/liter of 1 mg/ml stock in DMSO) and300 μl/liter Silwet L-77. Plants approximately 1 month old were dippedinto the media for 15 seconds, being sure to submerge the newestinflorescence. The plants were then laid down on their sides and covered(transparent or opaque) for 24 hours, then washed with water, and placedupright. The plants were grown at 22° C., with a 16-hour light/8-hourdark photoperiod. Approximately 4 weeks after dipping, the seeds wereharvested.

6.4—Selection of Transformed Plants.

Freshly harvested T₁ seed [AAD-13 (v1) gene] was allowed to dry for 7days at room temperature. T₁ seed was sown in 26.5×51-cm germinationtrays (T.O. Plastics Inc., Clearwater, Minn.), each receiving a 200 mgaliquots of stratified T₁ seed (˜10,000 seed) that had previously beensuspended in 40 ml of 0.1% agarose solution and stored at 4° C. for 2days to complete dormancy requirements and ensure synchronous seedgermination.

Sunshine Mix LP5 (Sun Gro Horticulture Inc., Bellevue, Wash.) wascovered with fine vermiculite and subirrigated with Hoagland's solutionuntil wet, then allowed to gravity drain. Each 40 ml aliquot ofstratified seed was sown evenly onto the vermiculite with a pipette andcovered with humidity domes (KORD Products, Bramalea, Ontario, Canada)for 4-5 days. Domes were removed 1 day prior to initial transformantselection using glufosinate postemergence spray (selecting for theco-transformed PAT gene).

Seven days after planting (DAP) and again 11 DAP, T₁ plants (cotyledonand 2-4-1f stage, respectively) were sprayed with a 0.2% solution ofLiberty herbicide (200 g ai/L glufosinate, Bayer Crop Sciences, KansasCity, Mo.) at a spray volume of 10 ml/tray (703 L/ha) using a DeVilbisscompressed air spray tip to deliver an effective rate of 280 g ai/haglufosinate per application. Survivors (plants actively growing) wereidentified 4-7 days after the final spraying and transplantedindividually into 3-inch pots prepared with potting media (Metro Mix360). Transplanted plants were covered with humidity domes for 3-4 daysand placed in a 22° C. growth chamber as before or moved to directly tothe greenhouse. Domes were subsequently removed and plants reared in thegreenhouse (22±5° C., 50±30% RH, 14 h light:10 dark, minimum 500 μE/m²s¹natural+supplemental light) at least 1 day prior to testing for theability of AAD-13 (v1) to provide phenoxy auxin herbicide resistance.

T₁ plants were then randomly assigned to various rates of 2,4-D. ForArabidopsis, 50 g ae/ha 2,4-D is an effective dose to distinguishsensitive plants from ones with meaningful levels of resistance.Elevated rates were also applied to determine relative levels ofresistance (280, 560, 1120, or 2240 g ae/ha). Tables 11 and 12 showcomparisons drawn to an aryloxyalkanoate herbicide resistance gene(AAD-12 (v1)) previously described in PCT/US2006/042133.

All auxin herbicide applications were made using the DeVilbiss sprayeras described above to apply 703 L/ha spray volume (0.4 mlsolution/3-inch pot) or applied by track sprayer in a 187 L/ha sprayvolume. 2,4-D used was either technical grade (Sigma, St. Louis, Mo.)dissolved in DMSO and diluted in water (<1% DMSO final concentration) orthe commercial dimethylamine salt formulation (456 g ae/L, NuFarm, StJoseph, Mo.). Dichlorprop used was commercial grade formulated aspotassium salt of R-dichlorprop (600 g ai/L, AH Marks). As herbiciderates increased beyond 800 g ae/ha, the pH of the spray solution becameexceedingly acidic, burning the leaves of young, tender Arabidopsisplants and complicating evaluation of the primary effects of theherbicides.

Some T₁ individuals were subjected to alternative commercial herbicidesinstead of a phenoxy auxin. One point of interest was determiningwhether the pyridyloxyacetate auxin herbicides, triclopyr andfluoroxypyr, could be effectively degraded in planta. Herbicides wereapplied to T₁ plants with use of a track sprayer in a 187 L/ha sprayvolume. T₁ plants that exhibited tolerance to 2,4-D DMA were furtheraccessed in the T₂ generation.

6.5—Results of Selection of Transformed Plants.

The first Arabidopsis transformations were conducted using AAD-13 (v1)(plant optimized gene). T₁ transformants were first selected from thebackground of untransformed seed using a glufosinate selection scheme.Over 160,000 T₁ seed were screened and 238 glufosinate resistant plantswere identified (PAT gene), equating to a transformation/selectionfrequency of 0.15% which lies in the normal range of selection frequencyof constructs where PAT+Liberty are used for selection. T₁ plantsselected above were subsequently transplanted to individual pots andsprayed with various rates of commercial aryloxyalkanoate herbicides.Table 11 compares the response of AAD-13 (v1) and control genes toimpart 2,4-D resistance to Arabidopsis T₁ transformants. Response ispresented in terms of % visual injury 2 WAT. Data are presented as ahistogram of individuals exhibiting little or no injury (<20%), moderateinjury (20-40%), or severe injury (>40%). An arithmetic mean andstandard deviation is presented for each treatment. The range inindividual response is also indicated in the last column for each rateand transformation. PAT/Cry1F-trans formed Arabidopsis served as anauxin-sensitive transformed control. The AAD-13 (v1) gene impartedherbicide resistance to individual T₁ Arabidopsis plants. Within a giventreatment, the level of plant response varied greatly and can beattributed to the fact each plant represents an independenttransformation event. Of important note, at each 2,4-D rate tested,there were individuals that were unaffected while some were severelyaffected. An overall population injury average by rate is presented inTable 11 simply to demonstrate the significant difference between theplants transformed with AAD-13 (v1) versus the AAD-12 (v1) orPAT/Cry1F-transformed controls. At high rates the spray solution becomeshighly acidic unless buffered therefore some of the injury may beattributed to the acidity of the spray solution. Arabidopsis grownmostly in the growth chamber has a very thin cuticle and severe burningeffects can complicate testing at these elevated rates. Nonetheless,many individuals have survived 2,240 g ae/ha 2,4-D with little or noinjury.

TABLE 11 AAD-13 (v1) transformed T1 Arabidopsis response to a range of2,4-D rates applied postemergence, compared to an AAD-12 v1 (T4)homozygous resistant population, or a Pat-Cry1F transformed,auxin-sensitive control (14 DAT). % Injury % Injury <20% 20-40% >40% AveStd dev Range (%) AAD-13 (v1) gene T₁ plants Averages   0 g ae/ha 2,4-DDMA 20 0 0 0 0 0  280 g ae/ha 2,4-D DMA 12 4 4 21 31 0-90  560 g ae/ha2,4-D DMA 17 2 0 2 6 0-20 1120 g ae/ha 2,4-D DMA 20 0 0 2 4 0-10 2240 gae/ha 2,4-D DMA 14 3 3 15 23 0-70 PAT/Cry1F (transformed control)Averages   0 g ae/ha 2,4-D DMA 20 0 0 0 0 0  280 g ae/ha 2,4-D DMA 0 020 100 0 100  560 g ae/ha 2,4-D DMA 0 0 20 100 0 100 1120 g ae/ha 2,4-DDMA 0 0 20 100 0 100 2240 g ae/ha 2,4-D DMA 0 0 20 100 0 100 HomozygousAAD-12 (v1) gene T₄ plants Averages   0 g ae/ha 2,4-D DMA 20 0 0 0 0 0 280 g ae/ha 2,4-D DMA 20 0 0 0 0 0  560 g ae/ha 2,4-D DMA 20 0 0 1 30-10 1120 g ae/ha 2,4-D DMA 20 0 0 2 4 0-15 2240 g ae/ha 2,4-D DMA 16 31 13 13 0-50

Table 12 shows a similarly conducted dose response of T₁ Arabidopsis tothe phenoxypropionic acid, dichlorprop. The data shows that theherbicidally active (R-) isomer of dichlorprop does not serve as asuitable substrate for AAD-13 (v1) or AAD-12 (v1). The fact that AAD-1(v3) will metabolize R-dichlorprop well enough to impart commerciallyacceptable tolerance is one distinguishing characteristic that separatesthe three genes (Table 12 and Example 7 of PCT/US2006/042133 (Wright etal., filed Oct. 27, 2006). AAD-1 and AAD-13 are considered R- andS-specific α-ketoglutarate dioxygenases, respectively.

TABLE 12 T1 Arabidopsis response to a range of R-dichlorprop ratesapplied postemergence. (14 DAT) % Injury % Injury <20% 20-40% >40% AveStd dev Range (%) AAD-13 (v1) gene T₁ plants Averages  0 g ae/ha 20 0 00 0 0 800 g ae/ha R-dichloroprop 0 0 20 100 0 100 Wildtype(untransformed control) Averages  0 g ae/ha 20 0 0 0 0 0 800 g ae/haR-dichloroprop 0 0 20 100 0 100 Homozygous AAD-12 (v1) gene T₄ plantsAverages  0 g ae/ha 20 0 0 0 0 0 800 g ae/ha R-dichloroprop 0 0 20 100 0100

6.6—AAD-13 (v1) as a Selectable Marker.

The ability to use AAD-13 (v1) as a selectable marker using 2,4-D as theselection agent will be was analyzed with Arabidopsis transformed asdescribed above. Approximately 50 T₄ generation Arabidopsis seed(homozygous for AAD-13 (v1)) will be spiked into approximately 5,000wildtype (sensitive) seed. Several treatments will be compared, eachtray of plants will receive either one or two application timings of2,4-D in one of the following treatment schemes: 7 DAP, 11 DAP, or 7followed by 11 DAP. Since all individuals also contain the PAT gene inthe same transformation vector, AAD-13 selected with 2,4-D can bedirectly compared to PAT selected with glufosinate.

Treatments will be applied with a DeVilbiss spray tip as previouslydescribed. Plants will be identified as Resistant or Sensitive 17 DAP.The optimum treatment of 75 g ae/ha 2,4-D applied 7 and 11 days afterplanting (DAP), is equally effective in selection frequency, and resultsin less herbicidal injury to the transformed individuals than theLiberty selection scheme. These results will indicate that AAD-13 (v1)can be effectively used as an alternative selectable marker for apopulation of transformed Arabidopsis.

6.7—Heritability.

A variety of T₁ events were self-pollinated to produce T₂ seed. Theseseed were progeny tested by applying Liberty (280 g ae/ha) to 100 randomT₂ siblings. Each individual T₂ plant was transplanted to 7.5-cm squarepots prior to spray application (track sprayer at 187 L/ha applicationsrate). Fifty percent of the T₁ families (T₂ plants) segregated in theanticipated 3 Resistant:1 Sensitive model for a dominantly inheritedsingle locus with Mendelian inheritance as determined by Chi squareanalysis (P>0.05).

Seed were collected from 12 to 20 T₂ individuals (T₃ seed). Twenty-fiveT₃ siblings from each of eight randomly-selected T₂ families wereprogeny tested as previously described. Half of the T₂ families testedwere homozygous (non-segregating populations) in each line. These datashow will show that AAD-13 (v1) is stably integrated and inherited in aMendelian fashion to at least three generations.

6.8—Additional Foliar Applications Herbicide Resistance in AAD-13Arabidopsis.

The ability of AAD-13 (v1) to provide resistance to otheraryloxyalkanoate auxin herbicides in transgenic Arabidopsis wasdetermined by foliar application of various substrates. T₂ generationArabidopsis seed was stratified, and sown into selection trays much likethat of Arabidopsis (Example 6.4). A transformed-control line containingPAT and the insect resistance gene Cry1F was planted in a similarmanner. Seedlings were transferred to individual 3-inch pots in thegreenhouse. All plants were sprayed with the use of a track sprayer setat 187 L/ha. The plants were sprayed with a range of pyridyloxyacetateherbicides: 200-800 g ae/ha triclopyr (Garlon 3A, Dow AgroSciences) and200-800 g ae/ha fluoroxypyr (Starane, Dow AgroSciences). The 2,4-Dmetabolite resulting from AAD-13 activity, 2,4-dichlorophenol (DCP,Sigma) (at a molar equivalent to 280-2240 g ae/ha of 2,4-D, technicalgrade will also be tested. All applications were formulated in water.Each treatment was replicated 3-4 times. Plants were evaluated at 3 and14 days after treatment.

AAD-13-transformed plants were also clearly protected from thefluoroxypyr herbicide injury that was seen in the transformed controlline, Pat/Cry1F (see Table 13); however, AAD-13-transformed plants wereseverely injured by triclopyr. These results confirm that AAD-13 (v1) inArabidopsis provides resistance to the pyridyloxyacetic auxins tested.The AAD-13 (v1) gene provided robust resistance up to 400 g ae/hafluoroxypyr, whereas the AAD-12 (v1) gene provided only modest level oftolerance as low as 200 g/ha. The AAD-13 (v1) gene providedsignificantly less tolerance to triclopyr than the AAD-12 (v1) gene. Thesignificantly greater tolerance to fluoroxypyr is unexpected and allowsdistinction of AAD-13 (v1)-type activity from AAD-12 (v1) and issupported by the enzymatic data of Example 5.

TABLE 13 Comparison of T2 AAD-13 (v1) and transformed controlArabidopsis plant response to various foliar-applied auxinic herbicides.Pyridyloxyacetic auxins Ave. % Injury 14DAT Segregating T2 AAD-13Homozygous (v1) plants T4 AAD-12 Pat/Cry1f- Herbicide Treatment(pDAB4114.01.094) (v1) plants Control 200 g ae/ha triclopyr 75 25 100400 g ae/ha triclopyr 90 33 100 800 g ae/ha triclopyr 100 79 100 200 gae/ha fluroxypyr 10 48 100 400 g ae/ha fluroxypyr 16 55 100 800 g ae/hafluroxypyr 55 60 100

Example 7 Transformation of Additional Crop Species

Corn may be transformed to provide high levels resistance to 2,4-D andfluoroxypyr by utilizing the same techniques previously described inExample #8 of WO 2007/053482 (PCT/US2006/042133 (Wright et al.).

Soybean may be transformed to provide high levels resistance to 2,4-Dand fluoroxypyr by utilizing the same techniques previously described inExample #11 or Example #13 of WO 2007/053482 (PCT/US2006/042133 (Wrightet al.)).

Cotton may be transformed to provide high levels resistance to 2,4-D andfluoroxypyr by utilizing the same techniques previously described inExamples #14 of patent application PCT/US2005/014737 (Wright et al.,filed May 2, 2005) or Example #12 of WO 2007/053482 (Wright et al.).

Canola may be transformed to provide high levels resistance to 2,4-D andfluoroxypyr by utilizing the same techniques previously described inExample #26 of patent application PCT/US2005/014737 (Wright et al.,filed May 2, 2005) or Example #22 of WO 2007/053482 (Wright et al.).

Example 8 Protein Detection from Transformed Plants Via Antibody

Antibodies and subsequent ELISA assays can be developed and implementedas described in Example 9 of WO 2007/053482 (Wright et al.), forexample.

Example 9 Tobacco Transformation

Tobacco transformation with Agrobacterium tumefaciens was carried out bya method similar, but not identical, to published methods (Horsch etal., 1988). To provide source tissue for the transformation, tobaccoseed (Nicotiana tabacum cv. KY160) was surface sterilized and planted onthe surface of TOB-medium, which is a hormone-free Murashige and Skoogmedium (Murashige and Skoog, 1962) solidified with agar. Plants weregrown for 6-8 weeks in a lighted incubator room at 28-30° C. and leavescollected sterilely for use in the transformation protocol. Pieces ofapproximately one square centimeter were sterilely cut from theseleaves, excluding the midrib. Cultures of the Agrobacterium strains(EHA101S containing pDAB3278, aka pDAS1580, AAD-13 (v1)+PAT), grownovernight in a flask on a shaker set at 250 rpm at 28° C., was pelletedin a centrifuge and resuspended in sterile Murashige & Skoog salts, andadjusted to a final optical density of 0.5 at 600 nm. Leaf pieces weredipped in this bacterial suspension for approximately 30 seconds, thenblotted dry on sterile paper towels and placed right side up on TOB+medium (Murashige and Skoog medium containing 1 mg/L indole acetic acidand 2.5 mg/L benzyladenine) and incubated in the dark at 28° C. Two dayslater the leaf pieces were moved to TOB+ medium containing 250 mg/Lcefotaxime (Agri-Bio, North Miami, Fla.) and 5 mg/L glufosinate ammonium(active ingredient in Basta, Bayer Crop Sciences) and incubated at28-30° C. in the light. Leaf pieces were moved to fresh TOB+ medium withcefotaxime and Basta twice per week for the first two weeks and once perweek thereafter. Four to six weeks after the leaf pieces were treatedwith the Agrobacteria; small plants arising from transformed foci wereremoved from this tissue preparation and planted into mediumTOB-containing 250 mg/L cefotaxime and 10 mg/L Basta in Phytatray™ IIvessels (Sigma). These plantlets were grown in a lighted incubator room.After 3 weeks, stem cuttings were taken and re-rooted in the same media.Plants were ready to send out to the greenhouse after 2-3 additionalweeks.

Plants were moved into the greenhouse by washing the agar from theroots, transplanting into soil in 13.75 cm square pots, placing the potinto a Ziploc® bag (SC Johnson & Son, Inc.), placing tap water into thebottom of the bag, and placing in indirect light in a 30° C. greenhousefor one week. After 3-7 days, the bag was opened; the plants werefertilized and allowed to grow in the open bag until the plants weregreenhouse-acclimated, at which time the bag is removed. Plants weregrown under ordinary warm greenhouse conditions (30° C., 16 hour day, 8hour night, minimum natural+supplemental light=500 μE/m²s¹).

Prior to propagation, T₀ plants were sampled for DNA analysis todetermine the insert copy number. The PAT gene which was molecularlylinked to AAD-13 (v1) was assayed for convenience. Fresh tissue wasplaced into tubes and lyophilized at 4° C. for 2 days. After the tissuewas fully dried, a tungsten bead (Valenite) was placed in the tube andthe samples were subjected to 1 minute of dry grinding using a Kelcobead mill. The standard DNeasy DNA isolation procedure was then followed(Qiagen, DNeasy 69109). An aliquot of the extracted DNA was then stainedwith Pico Green (Molecular Probes P7589) and read in the fluorometer(BioTek) with known standards to obtain the concentration in ng/μl.

The DNA samples were diluted to approximately 9 ng/μl and then denaturedby incubation in a thermocycler at 95° C. for 10 minutes. Signal Probemix is then prepared using the provided oligo mix and MgCl₂ (Third WaveTechnologies). An aliquot of 7.5 μl is placed in each well of theInvader assay plate followed by an aliquot of 7.5 μl of controls,standards, and 20 ng/μl diluted unknown samples. Each well was overlaidwith 15 μl of mineral oil (Sigma). The plates were incubated at 63° C.for 1.5 hours and read on the fluorometer (Biotek). Calculation of %signal over background for the target probe divided by the % signal overbackground internal control probe will calculate the ratio. The ratio ofknown copy standards developed and validated with southern blot analysiswas used to identify the estimated copy of the unknown events.

All events were also assayed for the presence of the AAD-13 (v1) gene byPCR using the same extracted DNA samples. A total of 100 ng of total DNAwas used as template. 20 mM of each primer was used with the Takara ExTaq PCR Polymerase kit. Primers for the Plant Transcription Unit (PTU)PCR AAD-13 were (SdpacodF: ATGGCTCA TGCTGCCCTCAGCC) (SEQ ID NO:6) and(SdpacodR: CGGGCAGGCCTAACTCCACC AA) (SEQ ID NO:7). The PCR reaction wascarried out in the 9700 Geneamp thermocycler (Applied Biosystems), bysubjecting the samples to 94° C. for 3 minutes and 35 cycles of 94° C.for 30 seconds, 64° C. for 30 seconds, and 72° C. for 1 minute and 45seconds followed by 72° C. for 10 minutes. PCR products were analyzed byelectrophoresis on a 1% agarose gel stained with EtBr.

9.1—Selection of Transformed Plants.

Following the acclimation in the greenhouse T₀ plants were then randomlyassigned to various rates of 2,4-D DMA ranging from 140 to 2240 g ae/haat 4-fold increments. For tobacco, 140 g ae/ha 2,4-D is an effectivedose to distinguish sensitive plants from ones with meaningful levels ofresistance. Table 14 shows comparisons drawn to T₀ plants transformedwith a glufosinate herbicide resistance gene (PAT/Cry1F-transformedtobacco). Data demonstrated that AAD-13 (v1) when transformed in tobaccoplants provides robust tolerance to 2,4-D DMA to at least 2240 g ae/ha.

TABLE 14 Comparison of T₀ AAD-13 (v1) and transformed (PAT) controltobacco plant respose to various rates of 2,4-D DMA 14 days afterapplication. % Injury % Injury <20% 20-40% >40% Ave Std dev Range (%)PAT/Cry1F (transformed controls) Averages   0 g ae/ha 2,4-D DMA 3 0 00.0 0.0 0  140 g ae/ha 2,4-D DMA 0 1 2 47.0 6.0 40-50   560 g ae/ha2,4-D DMA 0 0 3 75.0 0.0 75 2240 g ae/ha 2,4-D DMA 0 0 3 97.0 8.0 90-100AAD-13 (v1) gene T0 plants Averages   0 g ae/ha 2,4-D DMA 2 0 0 0.0 0.00  140 g ae/ha 2,4-D DMA 2 0 0 8.0 11.0 0-15  560 g ae/ha 2,4-D DMA 2 00 3.0 4.0 0-5  2240 g ae/ha 2,4-D DMA 2 0 0 5.0 0.0 5

T1 seed from individual T0 transformants were saved and seed wasstratified and sown onto selection trays in the greenhouse much likethat of Example 5 Prior to testing elevated rates of 2,4-D DMA, each T₁line were progeny tested by applying 2,4-D DMA (560 g ae/ha) to 100random T₁ siblings. Spray applications were made as previous describedwith a track sprayer calibrated to an application rate of 187 L/ha.Forty-three percent of the T₀ families (T₁ plants) segregated in theanticipated 3 Resistant:1 Sensitive model for a dominantly inheritedsingle locus with Mendelian inheritance as determined by Chi squareanalysis (P>0.05).

Seed were collected from 12 to 20 T₂ individuals (T₂ seed). Twenty-fiveT₃ siblings from each of eight randomly-selected T₂ families will beprogeny tested as previously described. Approximately one-third of theT₂ families are anticipated to be homozygous (non-segregatingpopulations) in each line. These data show will show that AAD-13 (v1) isstably integrated and inherited in a Mendelian fashion to at least threegenerations.

Surviving T₁ plants were then randomly assigned to various rates of2,4-D. For tobacco, 140 g ae/ha 2,4-D is an effective dose todistinguish sensitive plants from ones with meaningful levels ofresistance. Elevated rates were also applied to determine relativelevels of resistance (140, 560, or 2240 g ae/ha). Table 15 shows thecomparisons drawn to an untransformed control (KY160) variety oftobacco.

All auxin herbicide applications were applied by track sprayer in a 187L/ha spray volume. 2,4-D used was the commercial dimethylamine saltformulation (456 g ae/L, NuFarm, St Joseph, Mo.). Some T₁ individualswere subjected to alternative commercial herbicides instead of a phenoxyauxin. One point of interest was determining whether thepyridyloxyacetate auxin herbicides, triclopyr and fluoroxypyr, could beeffectively degraded in planta. Herbicides were applied to T₁ plantswith use of a track sprayer in a 187 L/ha spray volume. T₁ plants thatexhibited tolerance to 2,4-D DMA were further accessed in the T₂generation.

9.2—Results of Selection of Transformed Plants.

T₁ transformants were first selected from the background ofuntransformed plants using a 2,4-D selection scheme. Table 15 comparesthe response of AAD-13 (v1) and control genes to impart 2,4-D resistanceto tobacco T₁ transformants. Response is presented in terms of % visualinjury 2 WAT. Data are presented as a histogram of individualsexhibiting little or no injury (<20%), moderate injury (20-40%), orsevere injury (>40%). An arithmetic mean and standard deviation ispresented for each treatment. The range in individual response is alsoindicated in the last column for each rate and transformation. KY160untransformed tobacco served as an auxin-sensitive control. The AAD-13(v1) gene imparted herbicide resistance to individual T₁ tobacco plants.

TABLE 15 AAD-13 (v1) transformed T₁ tobacco response to a range of 2,4-Drates applied postemergence, compared to an untransformed,auxin-sensitive control. % Injury % Injury <20% 20-40% >40% Ave Std devRange (%) Wildtype (untransformed control) Averages Untreated control 30 0 0.0 0.0 0  140 g ae/ha 2,4-DMA 0 0 3 80.0 0.0 80   560 g ae/ha2,4-DMA 0 0 3 88.0 1.0 88-89 2240 g ae/ha 2,4-DMA 0 0 3 92.0 3.0 90-95AAD-13 (v1) gene T₁ plants Averages Untreated control 3 0 0 0.0 0.0 0 140 g ae/ha 2,4-DMA 3 0 0 0.0 0.0 0  560 g ae/ha 2,4-DMA 3 0 0 0.0 0.00 2240 g ae/ha 2,4-DMA 3 0 0 2.0 3.0 0-5

9.3—Additional Foliar Applications Herbicide Resistance in AAD-13Tobacco.

The ability of AAD-13 (v1) to provide resistance to otheraryloxyalkanoate auxin herbicides in transgenic tobacco was determinedby foliar application of various substrates. Extra T₁ generation plantsfollowing the T1 progeny testing were sprayed with the use of a tracksprayer set at 187 L/ha. The plants were sprayed with a range ofpyridyloxyacetate herbicides: 140-1120 g ae/ha triclopyr (Garlon 3A, DowAgroSciences) and 280-1120 g ae/ha fluoroxypyr (Starane, DowAgroSciences). All applications were formulated in water. Each treatmentwas replicated 3 times. Plants were evaluated at 3 and 14 days aftertreatment.

AAD-13-transformed plants were poorly protected from the triclopyr butwere well protected from fluoroxypyr herbicide injury that was seen inthe untransformed control line (see Table 16). These results confirmthat AAD-13 (v1) in tobacco provides resistance to certain selectedpyridyloxyacetic auxin tested. The AAD-13 (v1) gene providedsignificantly tolerance up to 1120 g ae/ha fluoroxypyr, whereas the geneprovided only modest level of tolerance to triclopyr as low as 280 g/ha.These data confirm that AAD-13 (v1) provides a selectivity bias towardfluoroxypyr over triclopyr of the pyridyloxy auxins in multiple species.This unexpected observation further distinguishes the AAD-13 (v1) genefrom other herbicide tolerance enzymes of similar mechanism and isobserved in multiple plant species.

TABLE 16 Comparison of T₁ AAD-13 (v1) and untransformed control tobaccoplant response to various foliar applied auxinic herbicides 14 daysafter application. Pyridyloxyacetic auxins Segregating T1 KY160 AAD-13(v1) plants (untransformed Herbicide Treatment (pDAB4114[1]003.006)control)  280 g ae/ha triclopyr 53.0 82.0  560 g ae/ha triclopyr 65.088.0 1120 g ae/ha triclopyr 75.0 92.0  280 g ae/ha fluroxypyr 7.0 100.0 560 g ae/ha fluroxypyr 25.0 100.0 1120 g ae/ha fluroxypyr 37.0 100.0

Example 10 AAD-13 (v1) in Canola and Transformation of Other Crops

10.1 Canola Transformation.

The AAD-13 (v1) gene conferring resistance to 2,4-D can be used totransform Brassica napus with Agrobacterium-mediated transformationusing PAT as a selectable marker.

Seeds can be surface-sterilized with 10% commercial bleach for 10minutes and rinsed 3 times with sterile distilled water. The seeds willbe placed on one half concentration of MS basal medium (Murashige andSkoog, 1962) and maintained under growth regime set at 25° C., and aphotoperiod of 16 hrs light/8 hrs dark.

Hypocotyl segments (3-5 mm) would be excised from 5-7 day old seedlingsand placed on callus induction medium K1D1 (MS medium with 1 mg/Lkinetin and 1 mg/L 2,4-D) for 3 days as pre-treatment. The segments willthen be transferred into a petri plate, treated with Agrobacterium Z707Sor LBA4404 strain containing pDAB3759. The Agrobacterium shall be grownovernight at 28° C. in the dark on a shaker at 150 rpm and subsequentlyre-suspended in the culture medium.

After 30 min treatment of the hypocotyl segments with Agrobacterium,these would be placed back on the callus induction medium for 3 days.Following co-cultivation, the segments will be placed on K1D1TC (callusinduction medium containing 250 mg/L Carbenicillin and 300 mg/LTimentin) for one week or two weeks of recovery. Alternately, thesegments would be placed directly on selection medium K1D1H1 (abovemedium with 1 mg/L Herbiace). Carbenicillin and Timentin antibioticswould be used to kill the Agrobacterium. The selection agent Herbiaceallows the growth of the transformed cells.

Callused hypocotyl segments would be placed on B3Z1H1 (MS medium, 3 mg/Lbenzylamino purine, 1 mg/L Zeatin, 0.5 gm/L MES [2-(N-morpholino) ethanesulfonic acid], 5 mg/L silver nitrate, 1 mg/L Herbiace, Carbenicillinand Timentin) shoot regeneration medium. After 2-3 weeks shootsregenerate and hypocotyl segments along with the shoots are transferredto B3Z1H3 medium (MS medium, 3 mg/L benzylamino purine, 1 mg/L Zeatin,0.5 gm/L MES [2-(N-morpholino) ethane sulfonic acid], 5 mg/L silvernitrate, 3 mg/L Herbiace, Carbenicillin and Timentin) for another 2-3weeks.

Shoots would be excised from the hypocotyl segments and transferred toshoot elongation medium MESH5 or MES10 (MS, 0.5 gm/L MES, 5 or 10 mg/LHerbiace, Carbenicillin, Timentin) for 2-4 weeks. The elongated shootsare cultured for root induction on MSI.1 (MS with 0.1 mg/L Indolebutyricacid). Once the plants are well established root system, these will betransplanted into soil. The plants are acclimated under controlledenvironmental conditions in the Conviron for 1-2 weeks before transferto the greenhouse.

10.2—Agrobacterium Transformation of Other Crops

In light of the subject disclosure, additional crops can be transformedaccording to the subject invention using techniques that are known inthe art. For Agrobacterium-mediated trans-formation of rye, see, e.g.,Popelka and Altpeter (2003)., see, e.g., Hinchee et al., 1988. ForAgrobacterium-mediated transformation of sorghum, see, e.g., Zhao etal., 2000. For Agrobacterium-mediated transformation of barley, see,e.g., Tingay et al., 1997. For Agrobacterium-mediated transformation ofwheat, see, e.g., Cheng et al., 1997. For Agrobacterium-mediatedtransformation of rice, see, e.g., Hiei et al., 1997.

The Latin names for these and other plants are given below. It should beclear that these and other (non-Agrobacterium) transformation techniquescan be used to transform AAD-13 (v1), for example, into these and otherplants, including but not limited to Maize (Zea mays), Wheat (Triticumspp.), Rice (Oryza spp. and Zizania spp.), Barley (Hordeum spp.), Cotton(Abroma augusta and Gossypium spp.), Soybean (Glycine max), Sugar andtable beets (Beta spp.), Sugar cane (Arenga pinnata), Tomato(Lycopersicon esculentum and other spp., Physalis ixocarpa, Solanumincanum and other spp., and Cyphomandra betacea), Potato (Solanumtubersoum), Sweet potato (Ipomoea betatas), Rye (Secale spp.), Peppers(Capsicum annum, sinense, and frutescens), Lettuce (Lactuca sativa,perennis, and pulchella), Cabbage (Brassica spp), Celery (Apiumgraveolens), Eggplant (Solanum melongena), Peanut (Arachis hypogea),Sorghum (all Sorghum species), Alfalfa (Medicago sativua), Carrot(Daucus carota), Beans (Phaseolus spp. and other genera), Oats (Avenasativa and strigosa), Peas (Pisum, Vigna, and Tetragonolobus spp.),Sunflower (Helianthus annuus), Squash (Cucurbita spp.), Cucumber(Cucumis sativa), Tobacco (Nicotiana spp.), Arabidopsis (Arabidopsisthaliana), Turfgrass (Lolium, Agrostis, Poa, Cynadon, and other genera),Clover (Tifolium), Vetch (Vicia). Such plants, with AAD-13 (v1) genes,for example, are included in the subject invention.

AAD-13 (v1) has the potential to increase the applicability of keyauxinic herbicides for in-season use in many deciduous and evergreentimber cropping systems. Triclopyr, 2,4-D, and/or fluoroxypyr resistanttimber species would increase the flexibility of over-the-top use ofthese herbicides without injury concerns. These species would include,but not limited to: Alder (Alnus spp.), ash (Fraxinus spp.), aspen andpoplar species (Populus spp.), beech (Fagus spp.), birch (Betula spp.),cherry (Prunus spp.), eucalyptus (Eucalyptus spp.), hickory (Caryaspp.), maple (Acer spp.), oak (Quercus spp), and pine (Pinus spp). Useof auxin resistance for the selective weed control in ornamental andfruit-bearing species is also within the scope of this invention.Examples could include, but not be limited to, rose (Rosa spp.), burningbush (Euonymus spp.), petunia (Petunia spp), begonia (Begonia spp.),rhododendron (Rhododendron spp), crabapple or apple (Malus spp.), pear(Pyrus spp.), peach (Prunus spp), and marigolds (Tagetes spp.).

Example 11 Further Evidence of Surprising Results: AAD-13 vs. AAD-2

Freshly harvested T₁ Arabidopsis seed transformed with a plant optimizedAAD-13 (v1) or native AAD-2 (v1) gene (see PCT/US2005/014737) wereplanted and selected for resistance to glufosinate as previouslydescribed Plants were then randomly assigned to various rates of 2,4-D(50-3200 g ae/ha). Herbicide applications were applied by track sprayerin a 187 L/ha spray volume. 2,4-D used was the commercial dimethylaminesalt formulation (456 g ae/L, NuFarm, St Joseph, Mo.) mixed in 200 mMTris buffer (pH 9.0) or 200 mM HEPES buffer (pH7.5).

AAD-13 (v1) and AAD-2 (v1) did provide detectable 2,4-D resistanceversus the transformed and untransformed control lines; however,individuals varied in their ability to impart 2,4-D resistance toindividual T₁ Arabidopsis plants. Surprisingly, AAD-2 (v1) and AAD-2(v2) transformants were far less resistant to 2,4-D than the AAD-13 (v1)gene, both from a frequency of highly tolerant plants as well as overallaverage injury. No plants transformed with AAD-2 (v1) survived 200 gae/ha 2,4-D relatively uninjured (<20% visual injury), and overallpopulation injury was about 83% (see PCT/US2005/014737). Conversely,AAD-13 (v1) had a population injury average of about 15% when treatedwith 2,240 g ae/ha 2,4-D (Table 11). Comparison of both AAD-13 and AAD-2plant optimized genes indicates a significant advantage for AAD-13 (v1)in planta.

These results are unexpected given that the in vitro comparison of AAD-2(v1) (see PCT/US2005/014737) and AAD-13 (v2) indicated both were highlyefficacious at degrading 2,4-D and both shared an S-type specificitywith respect to chiral aryloxyalkanoate substrates. AAD-2 (v1) isexpressed in individual T₁ plants to varying levels; however, littleprotection from 2,4-D injury is afforded by this expressed protein. Nosubstantial difference was evident in protein expression level (inplanta) for the native and plant optimized AAD-2 genes (seePCT/US2005/014737). These data corroborate earlier findings that makethe functional expression of AAD-13 (v1) in planta, and resultingherbicide resistance to 2,4-D and selected pyridyloxyacetate herbicides,is unexpected.

Example 12 Preplant Burndown Applications

This and the following Examples are specific examples of novel herbicideuses made possible by the subject AAD-13 invention.

Preplant burndown herbicide applications are intended to kill weeds thathave emerged over winter or early spring prior to planting a given crop.Typically these applications are applied in no-till or reduced tillagemanagement systems where physical removal of weeds is not completedprior to planting. An herbicide program, therefore, must control a verywide spectrum of broadleaf and grass weeds present at the time ofplanting. Glyphosate, gramoxone, and glufosinate are examples ofnon-selective, non-residual herbicides widely used for preplant burndownherbicide applications. Some weeds, however, are difficult to control atthis time of the season due to one or more of the following: inherentinsensitivity of the weed species or biotype to the herbicide,relatively large size of winter annual weeds, and cool weatherconditions limiting herbicide uptake and activity. Several herbicideoptions are available to tankmix with these herbicides to increasespectrum and activity on weeds where the non-selective herbicides areweak. An example would be 2,4-D tankmix applications with glyphosate toassist in the control of Conyza canadensis (horseweed). Glyphosate canbe used from 420 to 1680 g ae/ha, more typically 560 to 840 g ae/ha, forthe preplant burndown control of most weeds present; however, 280-1120 gae/ha of 2,4-D can be applied to aid in control of many broadleaf weedspecies (e.g., horseweed). 2,4-D is an herbicide of choice because it iseffective on a very wide range of broadleaf weeds, effective even at lowtemperatures, and extremely inexpensive. However, if the subsequent cropis a sensitive dicot crop, 2,4-D residues in the soil (althoughshort-lived) can negatively impact the crop. Soybeans are a sensitivecrop and require a minimum time period of 7 days (for 280 g ae/ha 2,4-Drate) to at least 30 days (for 2,4-D applications of 1120 g ae/ha) tooccur between burndown applications and planting. 2,4-D is prohibited asa burndown treatment prior to cotton planting (see federal labels, mostare available through CPR, 2005 or online at cdms.net/manuf/manuf.asp).With AAD-13 (v1) transformed cotton or soybeans, these crops should beable to survive 2,4-D residues in the soil from burndown applicationsapplied right up to and even after planting before emergence of thecrop. The increased flexibility and reduced cost of tankmix (orcommercial premix) partners will improve weed control options andincrease the robustness of burndown applications in important no-tilland reduced tillage situations. This example is one of many options thatwill be available. Those skilled in the art of weed control will note avariety of other applications including, but not limited togramoxone+2,4-D or glufosinate+2,4-D by utilizing products described infederal herbicide labels (CPR, 2005) and uses described in AgrilianceCrop Protection Guide (2005), as examples. Those skilled in the art willalso recognize that the above example can be applied to any2,4-D-sensitive (or other phenoxy auxin herbicide) crop that would beprotected by the AAD-13 (v1) gene if stably transformed. Likewise, theunique attributes of AAD-13 allowing degradation of fluoroxypyr increaseutility by allowing substitution or tank mixes of 35-560 g ae/hafluoroxypyr to increase spectrum and/or increase the ability to controlperennial or viney weed species.

Example 13 In-Crop Use of Auxin Herbicides in Soybeans, Cotton, andOther Dicot Crops Transformed Only with AAD-13 (v1)

AAD-13 (v1) can enable the use of phenoxy auxin herbicides (e.g., 2,4-Dand MCPA) and pyridyloxy auxins (fluoroxypyr) for the control of a widespectrum of broadleaf weeds directly in crops normally sensitive to2,4-D. Application of 2,4-D at 280 to 2240 g ae/ha would control mostbroadleaf weed species present in agronomic environments. Moretypically, 560-1120 g ae/ha is used. For fluoroxypyr, application rateswould typically range from 35-560 g ae/ha, more typically 70-280 ae/ha.

An advantage to this additional tool is the extremely low cost of thebroadleaf herbicide component and potential short-lived residual weedcontrol provided by higher rates of 2,4-D and fluoroxypyr when used athigher rates, whereas a non-residual herbicide like glyphosate wouldprovide no control of later germinating weeds. This tool also provides amechanism to combine herbicide modes of action with the convenience ofHTC as an integrated herbicide resistance and weed shift managementstrategy.

A further advantage this tool provides is the ability to tankmix broadspectrum broadleaf weed control herbicides (e.g., 2,4-D and fluoroxypyr)with commonly used residual weed control herbicides. These herbicidesare typically applied prior to or at planting, but often are lesseffective on emerged, established weeds that may exist in the fieldprior to planting. By extending the utility of these aryloxy auxinherbicides to include at-plant, preemergence, or pre-plant applications,the flexibility of residual weed control programs increases. One skilledin the art would recognize the residual herbicide program will differbased on the crop of interest, but typical programs would includeherbicides of the chloracetmide and dinitroaniline herbicide families,but also including herbicides such as clomazone, sulfentrazone, and avariety of ALS-inhibiting, PPO-inhibiting, and HPPD-inhibitingherbicides.

Further benefits could include tolerance to 2,4-D or fluoroxypyrrequired before planting following aryloxyacetic acid auxin herbicideapplication (see previous example); and fewer problems fromcontamination injury to dicot crops resulting from incompletely cleanedbulk tanks that had contained 2,4-D or fluoroxypyr. Dicamba,R-dhichlorprop, and many other herbicides can still be used for thesubsequent control of AAD-13 (v1)-transformed dicot crop volunteers.

Those skilled in the art will also recognize that the above example canbe applied to any 2,4-D-sensitive (or other aryloxy auxin herbicide)crop that would be protected by the AAD-13 (v1) gene if stablytransformed. One skilled in the art of weed control will now recognizethat use of various commercial phenoxy or pyridyloxy auxin herbicidesalone or in combination with an herbicide is enabled by AAD-13 (v1)transformation. Specific rates of other herbicides representative ofthese chemistries can be determined by the herbicide labels compiled inthe CPR (Crop Protection Reference) book or similar compilation or anycommercial or academic crop protection references such as the CropProtection Guide from Agriliance (2005). Each alternative herbicideenabled for use in HTCs by AAD-13 (v1), whether used alone, tank mixed,or sequentially, is considered within the scope of this invention.

Example 14 In-Crop Use of Phenoxy Auxin and Pyridyloxy Auxin Herbicidesin AAD-13 (v1) Only Transformed Corn, Rice, and Other Monocot Species

In an analogous fashion, transformation of grass species (such as, butnot limited to, corn, rice, wheat, barley, or turf and pasture grasses)with AAD-13 (v1) would allow the use of highly efficacious phenoxy andpyridyloxy auxins in crops where normally selectivity is not certain.Most grass species have a natural tolerance to auxinic herbicides suchas the phenoxy auxins (i.e., 2,4-D). However, a relatively low level ofcrop selectivity has resulted in diminished utility in these crops dueto a shortened window of application timing or unacceptable injury risk.AAD-13 (v1)-transformed monocot crops would, therefore, enable the useof a similar combination of treatments described for dicot crops such asthe application of 2,4-D at 280 to 2240 g ae/ha to control mostbroadleaf weed species. More typically, 560-1120 g ae/ha is used. Forfluoroxypyr, application rates would typically range from 35-560 gae/ha, more typically 70-280 ae/ha.

An advantage to this additional tool is the extremely low cost of thebroadleaf herbicide component and potential short-lived residual weedcontrol provided by higher rates of 2,4-D or fluoroxypyr. In contrast, anon-residual herbicide like glyphosate would provide no control oflater-germinating weeds. This tool would also provide a mechanism torotate herbicide modes of action with the convenience of HTC as anintegrated-herbicide-resistance and weed-shift-management strategy in aglyphosate tolerant crop/AAD-13 (v1) HTC combination strategy, whetherone rotates crops species or not.

A further advantage this tool provides is the ability to tankmix broadspectrum broadleaf weed control herbicides (e.g., 2,4-D and fluoroxypyr)with commonly used residual weed control herbicides. These herbicidesare typically applied prior to or at planting, but often are lesseffective on emerged, established weeds that may exist in the fieldprior to planting. By extending the utility of these aryloxy auxinherbicides to include at-plant, preemergence, or pre-plant applications,the flexibility of residual weed control programs increases. One skilledin the art would recognize the residual herbicide program will differbased on the crop of interest, but typical programs would includeherbicides of the chloracetmide and dinitroaniline herbicide families,but also including herbicides such as clomazone, sulfentrazone, and avariety of ALS-inhibiting, PPO-inhibiting, and HPPD-inhibitingherbicides.

The increased tolerance of corn, rice, and other monocots to the phenoxyor pyridyloxy auxins shall enable use of these herbicides in-cropwithout growth stage restrictions or the potential for crop leaning;unfurling phenomena such as “rat-tailing,” growth regulator-inducedstalk brittleness in corn, or deformed brace roots. Each alternativeherbicide enabled for use in HTCs by AAD-13 (v1), whether used alone,tank mixed, or sequentially, is considered within the scope of thisinvention.

Example 15 AAD-13 (v1) Stacked with Glyphosate Tolerance Trait in anyCrop

The vast majority of cotton, canola, corn, and soybean acres planted inNorth America contain a glyphosate tolerance (GT) trait, and adoption ofGT corn is on the rise. Additional GT crops (e.g., wheat, rice, sugarbeet, and turf) have been under development but have not beencommercially released to date. Many other glyphosate resistant speciesare in experimental to development stage (e.g., alfalfa, sugar cane,sunflower, beets, peas, carrot, cucumber, lettuce, onion, strawberry,tomato, and tobacco; forestry species like poplar and sweetgum; andhorticultural species like marigold, petunia, and begonias;isb.vt.edu/cfdocs/fieldtests1.cfm, 2005 on the World Wide Web). GTC'sare valuable tools for the sheer breadth of weeds controlled andconvenience and cost effectiveness provided by this system. However,glyphosate's utility as a now-standard base treatment is selecting forglyphosate resistant weeds. Furthermore, weeds that glyphosate isinherently less efficacious on are shifting to the predominant speciesin fields where glyphosate-only chemical programs are being practiced.By stacking AAD-13 (v1) with a GT trait, either through conventionalbreeding or jointly as a novel transformation event, weed controlefficacy, flexibility, and ability to manage weed shifts and herbicideresistance development could be improved. As mentioned in previousexamples, by transforming crops with AAD-13 (v1), monocot crops willhave a higher margin of phenoxy or pyridyloxy auxin safety, and phenoxyauxins can be selectively applied in dicot crops. Several scenarios forimproved weed control options can be envisioned where AAD-13 (v1) and aGT trait are stacked in any monocot or dicot crop species:

-   -   a) Glyphosate can be applied at a standard postemergent        application rate (420 to 2160 g ae/ha, preferably 560 to 840 g        ae/ha) for the control of most grass and broadleaf weed species.        For the control of glyphosate resistant broadleaf weeds like        Conyza canadensis or weeds inherently difficult to control with        glyphosate (e.g., Commelina spp, Ipomoea spp, etc), 280-2240 g        ae/ha (preferably 560-1120 g ae/ha) 2,4-D can be applied        sequentially, tank mixed, or as a premix with glyphosate to        provide effective control. For fluoroxypyr, application rates        would typically range from 35-560 g ae/ha, more typically 70-280        ae/ha.    -   b) Currently, glyphosate rates applied in GTC's generally range        from 560 to 2240 g ae/ha per application timing. Glyphosate is        far more efficacious on grass species than broadleaf weed        species. AAD-13 (v1)+GT stacked traits would allow        grass-effective rates of glyphosate (105-840 g ae/ha, more        preferably 210-420 g ae/ha). 2,4-D (at 280-2240 g ae/ha, more        preferably 560-1120 g ae/ha) could then be applied sequentially,        tank mixed, or as a premix with grass-effective rates of        glyphosate to provide necessary broadleaf weed control.        Fluoroxypyr at rates mentioned above would be acceptable        components in the treatment regimen. The low rate of glyphosate        would also provide some benefit to the broadleaf weed control;        however, primary control would be from the 2,4-D or fluoroxypyr.

One skilled in the art of weed control will recognize that use of one ormore commercial aryloxy auxin herbicides alone or in combination(sequentially or independently) is enabled by AAD-13 (v1) transformationinto crops. Specific rates of other herbicides representative of thesechemistries can be determined by the herbicide labels compiled in theCPR (Crop Protection Reference) book or similar compilation, labelscompiled online (e.g., cdms.net/manuf/manuf.asp), or any commercial oracademic crop protection guides such as the Crop Protection Guide fromAgriliance (2005). Each alternative herbicide enabled for use in HTCs byAAD-13 (v1), whether used alone, tank mixed, or sequentially, isconsidered within the scope of this invention.

Example 16 AAD-13 (v1) Stacked with Glufosinate Tolerance Trait in anyCrop

Glufosinate tolerance (PAT, bar) is currently present in a number ofcrops planted in North America either as a selectable marker for aninput trait like insect resistance proteins or specifically as an HTCtrait. Crops include, but are not limited to, glufosinate tolerantcanola, corn, and cotton. Additional glufosinate tolerant crops (e.g.,rice, sugar beet, soybeans, and turf) have been under development buthave not been commercially released to date. Glufosinate, likeglyphosate, is a relatively non-selective, broad spectrum grass andbroadleaf herbicide. Glufosinate's mode of action differs fromglyphosate. It is faster acting, resulting in desiccation and “burning”of treated leaves 24-48 hours after herbicide application. This isadvantageous for the appearance of rapid weed control. However, thisalso limits translocation of glufosinate to meristematic regions oftarget plants resulting in poorer weed control as evidenced by relativeweed control performance ratings of the two compounds in many species(Agriliance, 2005).

By stacking AAD-13 (v1) with a glufosinate tolerance trait, eitherthrough conventional breeding or jointly as a novel transformationevent, weed control efficacy, flexibility, and ability to manage weedshifts and herbicide resistance development could be improved. Severalscenarios for improved weed control options can be envisioned whereAAD-13 (v1) and a glufosinate tolerance trait are stacked in any monocotor dicot crop species:

-   -   a) Glufosinate can be applied at a standard postemergent        application rate (200 to 1700 g ae/ha, preferably 350 to 500 g        ae/ha) for the control of many grass and broadleaf weed species.        To date, no glufosinate-resistant weeds have been confirmed;        however, glufosinate has a greater number of weeds that are        inherently more tolerant than does glyphosate.        -   i) Inherently tolerant broadleaf weed species (e.g., Cirsium            arvensis Apocynum cannabinum, and Conyza candensis) could be            controlled by tank mixing 280-2240 g ae/ha, more preferably            560-2240 g ae/ha, 2,4-D for effective control of these more            difficult-to-control perennial species and to improve the            robustness of control on annual broadleaf weed species.            Fluoroxypyr would be acceptable components to consider in            the weed control regimen. For fluoroxypyr, application rates            would typically range from 35-560 g ae/ha, more typically            70-280 ae/ha.    -   b) A multiple combination of glufosinate (200-500 g        ae/ha)+/−2,4-D (280-1120 g ae/ha)+/−fluoroxypyr (at rates listed        above), for example, could provide more robust, overlapping weed        control spectrum. Additionally, the overlapping spectrum        provides an additional mechanism for the management or delay of        herbicide resistant weeds.

One skilled in the art of weed control will recognize that use of one ormore commercial aryloxyacetic auxin herbicides alone or in combination(sequentially or independently) is enabled by AAD-13 (v1) transformationinto crops. Specific rates of other herbicides representative of thesechemistries can be determined by the herbicide labels compiled in theCPR (Crop Protection Reference) book or similar compilation, labelscompiled online (e.g., cdms.net/manuf/manuf.asp), or any commercial oracademic crop protection guides such as the Crop Protection Guide fromAgriliance (2005). Each alternative herbicide enabled for use in HTCs byAAD-13 (v1), whether used alone, tank mixed, or sequentially, isconsidered within the scope of this invention.

The subject invention thus includes a transgenic plant (and plant cells)comprising an AAD-13 gene of the subject invention “stacked” with aDSM-2 gene of PCT/US2007/086813 (filed Dec. 7, 2007). Such DSM-2 genesinclude SEQ ID NOS:1 and 3 of that application. Those genes encodeproteins comprising SEQ ID NOS:2 and 4 of that application. Stillfurther, additional herbicide tolerance genes can be included inmultiple “stacks” comprising three or more such genes.

Example 17 AAD-13 (v1) Stacked with the AAD-1 (v3) Trait in any Crop

Homozygous AAD-13 (v1) and AAD-1 (v3) plants (see PCT/US2005/014737 forthe latter) can be both reciprocally crossed and F₁ seed collected. TheF₁ seed from two reciprocal crosses of each gene were stratified andtreated 4 reps of each cross were treated under the same spray regimenas used for the other testing with one of the following treatments: 70,140, 280 g ae/ha fluoroxypyr (selective for the AAD-12 (v1) gene); 280,560, 1120 g ae/ha R-dichloroprop (selective for the AAD-1 (v3) gene); or560, 1120, 2240 g ae/ha 2,4-D DMA (to confirm 2,4-D tolerance).Homozygous T₂ plants of each gene were also planted for use as controls.Plants were graded at 3 and 14 DAT. Spray results are shown in Table 24.

The results confirm AAD-13 (v1) can be successfully stacked with AAD-1(v3), thus increasing the spectrum herbicides that may be applied to thecrop of interest (phenoxyactetic acids+phenoxypropionic acids vspenoxyacetic acids+pyridyloxyacetic acids for AAD-1 and AAD-13,respectively). The complementary nature of herbicide cross resistancepatterns allows convenient use of these two genes as complementary andstackable field-selectable markers. In crops where tolerance with asingle gene may be marginal, one skilled in the art recognizes that onecan increase tolerance by stacking a second tolerance gene for the sameherbicide. Such can be done using the same gene with the same ordifferent promoters; however, as observed here, stacking and trackingtwo complementary traits can be facilitated by the distinguishing crossprotection to phenoxypropionic acids [from AAD-1 (v3)] orpyidyloxyacetic acids [AAD-13 (v1)].

The subject invention thus includes a transgenic plant (and plant cells)comprising an AAD-13 gene of the subject invention “stacked” with anAAD-1 gene of WO 2005/107437 (published Nov. 17, 2005; PCT/US2005/014737(filed May 2, 2005)). Such AAD-1 genes include SEQ ID NOS:3, 4, 5, and12 of that application. These genes encode proteins comprising SEQ IDNOS:9, 10, 11, and 13 of that application. Still further, additionalherbicide tolerance genes can be included in multiple “stacks”comprising three or more such genes.

Example 18 AAD-13 (v1) Stacked with the AAD-12 (v1) Trait in any Crop

Homozygous AAD-13 (v1) and AAD-12 (v1) plants (see WO 2007/053482 forthe latter) can be crossed and F₁ seed was collected. The F₁ seed fromtwo reciprocal crosses of each gene can be sown and F1 plants treatedunder the same spray regimen as used for the other testing with one ofthe following treatments: 70, 280, 1120 g ae/ha fluoroxypyr (selectivefor the AAD-12 (v1) gene); 70, 280, 1120 g ae/ha triclopyr (selectivefor the AAD-13 (v1) gene); or 560, 1120, 2240 g ae/ha 2,4-D DMA (toconfirm 2,4-D tolerance).

AAD-13 (v1) can be stacked with AAD-12 (v1), thus increasing thespectrum herbicides that may be applied to the crop of interest(phenoxyactetic acids+triclopyr vs phenoxyacetic acids+fluoroxypyr forAAD-12 and AAD-13, respectively). The complementary nature of herbicidecross resistance patterns allows convenient use of these two genes ascomplementary and stackable field-selectable markers. In crops wheretolerance with a single gene may be marginal, one skilled in the artrecognizes that one can increase tolerance by stacking a secondtolerance gene for the same herbicide. Such can be done using the samegene with the same or different promoters; however, as observed here,stacking and tracking two complementary traits can be facilitated by thedistinguishing cross protection to fluoroxypyr [from AAD-13 (v1)] andtriclopyr [AAD-12 (v1)].

The subject invention thus includes a transgenic plant (and plant cells)comprising an AAD-13 gene of the subject invention “stacked” with anAAD-12 gene of WO 2007/053482 (published May 10, 2007; PCT/US2006/042133(filed Oct. 27, 2006)). Such AAD-12 genes include SEQ ID NOS:1, 3, and 5of that application. Those genes encode proteins comprising SEQ ID NOS:2and 4 of that application. Still further, additional herbicide tolerancegenes can be included in multiple “stacks” comprising three or more suchgenes.

Example 19 AAD-13 (v1) Stacked with AHAS Trait in any Crop

Imidazolinone herbicide tolerance (AHAS, et al.) is currently present ina number of crops planted in North America including, but not limitedto, corn, rice, and wheat. Additional imidazolinone tolerant crops(e.g., cotton and sugar beet) have been under development but have notbeen commercially released to date. Many imidazolinone herbicides (e.g.,imazamox, imazethapyr, imazaquin, and imazapic) are currently usedselectively in various conventional crops. The use of imazethapyr,imazamox, and the non-selective imazapyr has been enabled throughimidazolinone tolerance traits like AHAS et al. This chemistry classalso has significant soil residual activity, thus being able to provideweed control extended beyond the application timing, unlike glyphosateor glufosinate-based systems. However, the spectrum of weeds controlledby imidazolinone herbicides is not as broad as glyphosate (Agriliance,2005). Additionally, imidazolinone herbicides have a mode of action(inhibition of acetolactate synthase, ALS) to which many weeds havedeveloped resistance (Heap, 2007). By stacking AAD-13 (v1) with animidazolinone tolerance trait, either through conventional breeding orjointly as a novel transformation event, weed control efficacy,flexibility, and ability to manage weed shifts and herbicide resistancedevelopment could be improved. As mentioned in previous examples, bytransforming crops with AAD-13 (v1), monocot crops will have a highermargin of phenoxy or pyridyloxy auxin safety, and these auxins can beselectively applied in dicot crops. Several scenarios for improved weedcontrol options can be envisioned where AAD-13 (v1) and an imidazolinonetolerance trait are stacked in any monocot or dicot crop species:

-   -   a) Imazethapyr can be applied at a standard postemergent        application rate of (35 to 280 g ae/ha, preferably 70-140 g        ae/ha) for the control of many grass and broadleaf weed species.        -   i) ALS-inhibitor resistant broadleaf weeds like Amaranthus            rudis, Ambrosia trifida, Chenopodium album (among others,            Heap, 2005) could be controlled by tank mixing 280-2240 g            ae/ha, more preferably 560-1120 g ae/ha, 2,4-D. For            fluoroxypyr, application rates would typically range from            35-560 g ae/ha, more typically 70-280 ae/ha.        -   ii) Inherently more tolerant broadleaf species to            imidazolinone herbicides like Ipomoea spp. can also be            controlled by tank mixing 280-2240 g ae/ha, more preferably            560-1120 g ae/ha, 2,4-D. See rates above for triclopyr or            fluoroxypyr.    -   b) A multiple combination of imazethapyr (35 to 280 g ae/ha,        preferably 70-140 g ae/ha)+/−2,4-D (280-1120 g        ae/ha)+/−fluoroxypyr (at rates listed above), for example, could        provide more robust, overlapping weed control spectrum.        Additionally, the overlapping spectrum provides an additional        mechanism for the management or delay of herbicide resistant        weeds.

One skilled in the art of weed control will recognize that use of any ofvarious commercial imidazolinone herbicides, phenoxyacetic orpyridyloxyacetic auxin herbicides, alone or in multiple combinations, isenabled by AAD-13 (v1) transformation and stacking with anyimidazolinone tolerance trait either by conventional breeding or geneticengineering. Specific rates of other herbicides representative of thesechemistries can be determined by the herbicide labels compiled in theCPR (Crop Protection Reference) book or similar compilation, labelscompiled online (e.g., cdms.net/manuf/manuf.asp), or any commercial oracademic crop protection guides such as the Crop Protection Guide fromAgriliance (2005). Each alternative herbicide enabled for use in HTCs byAAD-13 (v1), whether used alone, tank mixed, or sequentially, isconsidered within the scope of this invention.

Example 20 AAD-13 (v1) Stacked with Insect Resistance (IR) or OtherInput Traits in any Crop

Insect resistance in crops supplied by a transgenic trait is prevalentin corn and cotton production in North America and across the globe.Commercial products having combined IR and HT traits have been developedby multiple seed companies. These include Bt IR traits (e.g. Bt toxinslisted at the website lifesci.sussex.ac.uk, 2006) and any or all of theHTC traits mentioned above. The value this offering brings is theability to control multiple pest problems through genetic means in asingle offering. The convenience of this offering will be restricted ifweed control and insect control are accomplished independent of eachother. AAD-13 (v1) alone or stacked with one or more additional HTCtraits can be stacked with one or more additional input traits (e.g.,insect resistance, fungal resistance, or stress tolerance, et(isb.vt.edu/cfdocs/fieldtests1.cfm, 2005) either through conventionalbreeding or jointly as a novel transformation event. Benefits includethe convenience and flexibility described in previous examples togetherwith the ability to manage insect pests and/or other agronomic stressesin addition to the improved weed control offered by AAD-13 andassociated herbicide tolerance. Thus, the subject invention can be usedto provide a complete agronomic package of improved crop quality withthe ability to flexibly and cost effectively control any number ofagronomic issues.

Combined traits of IR and HT have application in most agronomic andhorticultural/ornamental crops and forestry. The combination of AAD-13and its commensurate herbicide tolerance and insect resistance affordedby any of the number of Bt or non-Bt IR genes are can be applied to thecrop species listed (but not limited to) in Example 13. One skilled inthe art of weed control will recognize that use of any of variouscommercial herbicides described in Examples 18-20, phenoxyacetic orpyridyloxyacetic auxin herbicides, alone or in multiple combinations, isenabled by AAD-13 (v1) transformation and stacking with thecorresponding HT trait or IR trait either by conventional breeding orgenetic engineering. Specific rates of other herbicides representativeof these chemistries can be determined by the herbicide labels compiledin the CPR (Crop Protection Reference) book or similar compilation,labels compiled online (e.g., cdms.net/manuf/manuf.asp), or anycommercial or academic crop protection guides such as the CropProtection Guide from Agriliance (2005). Each alternative herbicideenabled for use in HTCs by AAD-13 (v1), whether used alone, tank mixed,or sequentially, is considered within the scope of this invention.

Example 21 AAD-13 (v1) as an In Vitro Dicot Selectable Marker

Genetic engineering of plant cell, tissue, organ, and plant or organellesuch as plastid starts with the process of inserting genes of interestinto plant cells using a suitable delivery method. However, when a geneis delivered to plant cells, only an extremely small percentage of cellsintegrate the heterogeneous gene into their genome. In order to selectthose few cells that have incorporated the gene of interest, researcherslink a selectable or screenable “marker gene” to the gene of interest(GOI) in the vector. Cells that contain these markers are identifiedfrom the whole population of cells/tissue to which the DNA plasmidvector was delivered. By selecting those cells that express the markergene, researchers are able to identify those few cells that may haveincorporated the GOI into their genome. AAD-13 (v1) can function as aselectable marker when used as in Example #24 of patent application WO2007/053482 (Wright et al.).

1. An isolated polynucleotide that encodes a protein that enzymaticallydegrades an aryloxyalkanoate chemical substructure of anaryloxyalkanoate herbicide, wherein said polynucleotide is operablylinked to a promoter that is functional in a plant cell, and whereinsaid protein is at least 95% identical to a sequence selected from thegroup consisting of SEQ ID NO:2 and SEQ ID NO:4.
 2. The polynucleotideof claim 1, wherein said polynucleotide is at least 95% identical to asequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3,and SEQ ID NO:5.
 3. A plant cell comprising a polynucleotide of claim 2.4. A plant comprising a plurality of plant cells according to claim 3.5. The plant of claim 4, wherein said plant is a dicot.
 6. The plant ofclaim 4, wherein said plant is a soybean plant.
 7. The polynucleotide ofclaim 1, wherein said polynucleotide comprises a non-native codoncomposition having a bias towards plant codon usage to increaseexpression of said polynucleotide in a plant.
 8. The polynucleotide ofclaim 7, wherein said codon composition is biased toward dicot plantcodon usage.
 9. The polynucleotide of claim 7, wherein said promoter isa plant promoter.
 10. The polynucleotide of claim 7, wherein saidpromoter is a plant virus promoter.
 11. A method of controlling weeds inan area, said method comprising planting seeds in soil of the area,wherein said seeds comprise a polynucleotide that encodes a protein thatenzymatically degrades an aryloxyalkanoate chemical substructure of anaryloxyalkanoate herbicide, wherein said polynucleotide is operablylinked to a promoter that is functional in a plant cell; said methodfurther comprising applying said aryloxyalkanoate herbicide to saidarea; and wherein said protein is at least 95% identical to a sequenceselected from the group consisting of SEQ ID NO:2 and SEQ ID NO:4. 12.The method of claim 11, wherein said polynucleotide is at least 95%identical to a sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:3, and SEQ ID NO:5.
 13. The method of claim 11, whereinsaid seeds comprise a second polynucleotide that encodes a secondprotein that enzymatically degrades a second herbicide, and said methodcomprises applying said second herbicide to said area.
 14. The method ofclaim 13, wherein said seeds comprise a third polynucleotide thatencodes a third protein that enzymatically degrades a third herbicide,and said method comprises applying said third herbicide to said area.15. The method of claim 14, wherein said third herbicide is selectedfrom the group consisting of glufosinate and dicamba.
 16. The method ofclaim 13, wherein said second herbicide is glyphosate.
 17. The method ofclaim 13, wherein said aryloxyalkanoate herbicide is 2,4-D and saidsecond herbicide is glyphosate.
 18. The method of claim 17, wherein said2,4-D and said glyphosate are applied from a tank mix.
 19. The method ofclaim 11, wherein said herbicide is 2,4-D.
 20. The method of claim 11,wherein said seeds are seeds of a crop plant.
 21. The method of claim20, wherein said plant is a dicot.
 22. The method of claim 21, whereinsaid plant is a soybean plant.
 23. The method of claim 11, wherein saidaryloxyalkanoate herbicide is selected from the group consisting of (a)a phenoxyacetate or phenoxyacetic acid herbicide; (b) a phenoxypropionicacid herbicide; (c) a pyridyloxyalkanoic acid herbicide; and (d) anacid, salt, or ester form of an active ingredient of said herbicide. 24.The method of claim 23, wherein said pyridyloxyalkanoic acid herbicideis a pyridyloxyacetic acid herbicide.
 25. The method of claim 24,wherein said pyridyloxyacetic acid herbicide is selected from the groupconsisting of triclopyr and fluroxypyr.
 26. The method of claim 23,wherein (a) said phenoxyacetic acid herbicide is selected from the groupconsisting of 2,4-D and MCPA; and (b) said phenoxypropionic acidherbicide is selected from the group consisting of dichlorprop,mecoprop, an enantiomer of dichlorprop, and an enantiomer of mecoprop.27. The method of claim 11, wherein a phenoxyacetate herbicide and apyridyloxyacetate herbicide are applied to said area.
 28. The method ofclaim 11, wherein said method comprises growing crop plants, from saidseeds, in said area.